BACTERIA ENGINEERED TO TREAT METABOLIC DISEASES

Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of attenuating metabolic diseases are disclosed.

Description

BACKGROUND

Compositions and therapeutic methods for treating metabolic diseases are provided. In certain aspects, the compositions of the invention comprise bacteria that are genetically engineered to treat, modulate, and/or ameliorate metabolic diseases, particularly in low-oxygen environments, such as in the mammalian gut. In certain aspects, the compositions and methods of the invention as disclosed herein may be used for treating metabolic diseases such as obesity and type 2 diabetes. Obesity is caused by an imbalance between energy intake and expenditure, leading to the accumulation of unused energy in the form of fat. The World Health Organization considers obesity to be a global epidemic, and the United States Centers for Disease Control and Prevention estimates that nearly one third of adult Americans are obese. Diet and exercise may help reduce obesity and its associated pathologies, but adherence to a strict diet and exercise regime is challenging. Obesity may also be caused by other factors, e.g., mutations in genes regulating metabolic pathways (e.g., satiety, fatty acid oxidation, and mitochondrial function), which can contribute to energy imbalance. For example, congenital deficits in the signaling pathways for leptin, a satiety hormone, are known to cause obesity in humans and animal models.

Patients suffering from obesity are at increased risk of developing adverse physiological conditions, e.g., non-alcoholic fatty liver, cardiovascular diseases, type 2 diabetes mellitus (T2DM). The incidence of T2DM has increased 300% in the last three decades in the United States. T2DM patients are resistant to the effects of insulin, a hormone that regulates blood glucose levels, and frequently experience hyperglycemia, a condition in which blood glucose is above physiologically tolerable levels. When left untreated, hyperglycemia can result in severe complications such as hypertension, cardiovascular disease, inflammatory disease, blood vessel damage, nerve damage, cancer, and diabetes-induced coma.

T2DM involves the dysregulation of multiple metabolic organs, such as the pancreas, liver, skeletal muscle, adipose tissue, and brain, and it has been challenging to design therapeutics that target multiple tissue while avoiding systemic side effects. Insulin has been the first-line treatment for T2DM for decades. However, patients with severe T2DM may not respond to the insulin as a result of chronic insulin resistance. In addition, insulin must be administered multiple times throughout the day, which can adversely affect quality of life. Multiple therapies have been developed to treat T2DM, but not without limitations and sometimes life-threatening side effects. For example, thiazolidinedione was once widely used in order to increase the glucose metabolism in patients. However, the compound has been pulled from certain markets due to an increased association with heart failure (Nissen et al., 2007). Likewise, inhibitors of dipeptidyl peptidase-4 (DPP-4) have shown therapeutic promise, but may be linked to increased risk of pancreatic diseases (Karagiannis, et al., 2014).

Recently, researchers have demonstrated the close relationship between gut bacteria and metabolic disease (Harley et al., 2012). In obese mice, the ratio of firmicutes to bacteroidetes bacteria is increased (Harley et al., 2012; Mathur et al., 2015). These bacteria extract different amounts of energy from food, which may contribute to changes in energy balance. Similar changes have been also been observed in human studies (Harley et al., 2012; Mathur et al., 2015). Several molecules that are produced by gut bacteria have been shown to be metabolic regulators. For example, gut bacteria digest and break down dietary fiber into molecules such as acetate, butyrate, and propionate. These molecules are absorbed through intestinal cells, transferred to organs such as the liver and brain, and produce physiological changes, such as de novo glucose production and lipid synthesis (Brüssow et al., 2014; De Vadder et al., 2014; Lin et al., 2012). There has been an effort to engineer bacteria that produce N-acylphosphatidylethanolamines (NAPEs) (Chen et al., 2014). However, these bacteria express NAPEs constitutively and systemically, and NAPEs may be capable of “displac[ing] cholesterol from raft-like structures [and] may have dramatic implications for neural cell membrane function during stress and injury” (Terova et al., 2005). Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for metabolic diseases, including obesity and T2DM.

SUMMARY

The disclosure provides genetically engineered bacteria that are capable of treating metabolic diseases, including but not limited to, type 2 diabetes, obesity-related symptoms, Nonalcoholic Steatohepatitis (NASH), Prader Willi Syndrome, and cardiovascular disorders. The genetically engineered bacteria comprise one or more gene(s) or gene cassette(s), for the production of molecules which, inter alia, act as metabolic and/or satiety effectors and/or modulators of the inflammatory status and/or are able convert excess bile salts into non-toxic molecules, as described herein.

Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of metabolite consumption, or production of certain metabolites. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with metabolic disorders.

In some embodiments, the invention provides genetically engineered bacteria that are capable of producing one or more metabolic and/or satiety effector molecule(s), and/or one or more modulator(s) of inflammation, and/or one or more molecule(s) which reduces excess bile salt levels, and/or combinations thereof. In some embodiments, the invention provides genetically engineered bacteria that are capable of producing one or more metabolic and/or satiety effector molecule(s), and/or one or more modulator(s) of inflammation, and/or one or more molecule(s) which reduces excess bile salt levels, and/or combinations thereof, particularly in low-oxygen environments, e.g., the gut. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to treat metabolic diseases. In certain embodiments, the metabolic and/or satiety effector molecule and/or modulator of inflammation or/and or effector of excess bile salt reduction is stably produced by the genetically engineered bacteria, and/or the genetically engineered bacteria are stably maintained in vivo and/or in vitro. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating metabolic diseases.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non-native component(s), which mediate one or more mechanisms of action. The genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted.

In some embodiments, the genetically engineered bacteria comprise one or more of the following: (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of tryptophan, as described herein; (6) one or more genes or gene cassettes for the production of a tryptophan metabolite, including an indole and/or indole metabolite, as described herein; (7) one or more genes for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein; (8) one or more polypetides for secretion, including but not limited to secretion of GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthetic and/or catabolic enzymes of the tryptophan degradation pathways, and/or short chain fatty acid synthesis enzymes, in wild type or in mutated form (for increased stability or metabolic activity); (9) one or more components of secretion machinery, as described herein (10) one or more auxotrophies, e.g., deltaThyA; (11) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance; (12) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g. mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein; and (13) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway.

These gene(s)/gene cassette(s) may be under the control of constitutive or inducible promoters. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by molecules or metabolites indicative of liver damage (e.g., bilirubin) and/or metabolic disease, promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present in the gut, e.g., arabinose and tetracycline and othere described herein (e.g., metabolites not naturally present in the gut can be exogenously added). These gene(s)/gene cassette(s) may be under the control of constitutive and/or inducible promoters which are active or induced under in vitro conditions, e.g., during bacterial growth in a flask or other appropriate vessel for bacterial expansion, production, and/or manufacture, as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of an E. coli that is genetically engineered to express a kynurenine biosynthesis cassette and/or a tryptophan biosynthesis cassette and/or tryptophan catabolic cassette which produces bioactive tryptophan metabolites described herein and/or GLP-1 and/or a propionate gene cassette and/or a butyrate gene cassette under the control of a FNR-responsive promoter and further comprising a secretion system and a metabolite transporter system.

FIG. 2A depicts a metabolic pathway for butyrate production FIGS. 2B and 2C depict two schematics of two different butyrate producing circuits (found in SYN-503 and SYN-504), both under the control of a tetracycline inducible promoter. FIG. 2D depicts a schematic of a third butyrate gene cassette (found in SYN-505) under the control of a tetracycline inducible promoter. SYN-503 comprises a bdc2 butyrate cassette under control of tet promoter on a plasmid. A “bdc2 cassette” or “bdc2 butyrate cassette” refres to a butyrate producing cassette that comprises at least the following genes: bcd2, etfB3, etfA3, hbd, crt2, pbt, and buk genes. SYN-504 comprises a ter butyrate cassette (ter gene replaces the bcd2, etfB3, and etfA3 genes) under control of tet promoter on a plasmid. A “ter cassette” or “ter butyrate cassette” refers to a butyrate producing cassette that comprises at least the following genes: ter, thiA1, hbd, crt2, pbt, buk. SYN-505 comprises a tesB butyrate cassette (ter gene is present and tesB gene replaces the pbt gene and the buk gene) under control of tet promoter on a plasmid. A “tes or tesB cassette or “tes or tesB butyrate cassette” refers to a butyrate producing cassette that comprises at least ter, thiA1, hbd, crt2, and tesB genes. An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiA1, hbd, crt2, and tesB genes. In some embodiments, the tes or tesB cassette is under control of an inducible promoter other than tetracycline. Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

FIG. 3 depicts the gene organization of exemplary engineered bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 3A and 3B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 3A depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 3B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 3C and 3D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 3C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 3D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 3E and F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H202. In FIG. 3E, in the absence of H2O2, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 3F, in the presence of H2O2, the OxyR transcription factor interacts with H2O2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 4 depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 4A and 4B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (0₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 4B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 4C and 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 4C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 4D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 4E and 4F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H₂O₂. In FIG. 4E, in the absence of H₂O₂, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIGS. 4F, in the presence of H₂O₂, the OxyR transcription factor interacts with H₂O₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 5 depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 5A and 5B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, and tesB; black boxes) is expressed. FIG. 5B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 5C and 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 5C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) is expressed. In FIG. 5D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 5E and 5F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H₂O₂. In FIG. 5E, in the absence of H₂O₂, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) is expressed. In FIGS. 5F, in the presence of H₂O₂, the OxyR transcription factor interacts with H₂O₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 6 depicts a graph of butyrate production using the circuits shown in FIG. 48. Cells were grown in M9 minimal media containing 0.2% glucose and induced with ATC at early log phase. As seen in FIG. 6A, similar amounts of butyrate were produced for each construct under aerobic vs anaerobic conditions. The ter strain produces more butyrate overall. pLogic031 comprises (bdc2 butyrate cassette under control of tet promoter on a plasmid) and pLogic046 comprises (ter butyrate cassette under control of tet promoter on a plasmid). FIG. 6B depicts butyrate production of pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid)) and a Nissle strain comprising plasmid pLOGIC046-delta pbt.buk/tesB+, an ATC-inducible ter-comprising butyrate construct with a deletion in the pbt-buk genes and their replacement with the tesB gene. The tesB construct results in greater butyrate production.

FIG. 7 depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are SYN-503, SYN-504, SYN-510 (SYN-510 is the same as SYN-503 except that it further comprises a nuoB deletion), and SYN-511 (SYN-511 is the same as SYN-504 except that it further comprises a nuoB deletion). The NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.

FIG. 8A depicts a schematic of a butyrate producing circuit under the control of an FNR promoter. FIG. 8B depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-501 led to significant butyrate production under anaerobic conditions.

FIG. 9 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct or the pLogic046-nsrR-norB-butyrate construct, which produce more butyrate as compared to wild-type Nissle.

FIG. 10 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H2O, 100 mM butyrate in H20, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H2O2O (+) 200 mM butyrate.

FIG. 11 depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid cpopies. Integrated butyrate strains, SYN1001 and SYN1002 gave comparable butyrate production to the plasmid strain SYN501.

FIG. 12 depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.

FIG. 13 depicts a schematic illustrating a strategy for increasing butyrate and acetate production in engineered bacteria. Aerobic metabolism through the citric acid cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of the colon. E. coli makes high levels of acetate as an end production of fermentation. To improve acetate production, while still maintaining highlevels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of competing routes (shown in in rounded boxes) are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions of interest therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

FIG. 14A and FIG. 14B depict bar graphs showing Acetate/Butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 14A) and Acetate/Butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 14B). Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes.

FIG. 15A and FIG. 15B depicts the gene organization of an exemplary engineered bacterium of the invention and its induction under low-oxygen conditions for the production of propionate. FIG. 15A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (pct, lcdA, lcdB, lcdC, e0, acrB, acrC; black boxes) are expressed. FIG. 15B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.

FIG. 16 depicts an exemplary propionate biosynthesis gene cassette.

FIG. 17A, FIG. 17B and FIG. 17C depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate. FIG. 17A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd; black boxes) are expressed. FIG. 17B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. FIG. 17C depicts an exemplary propionate biosynthesis gene cassette.

FIG. 18A, FIG. 18B and FIG. 18C depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate. FIG. 18A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd, tesB; black boxes) are expressed. FIG. 18B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.

FIG. 19 depicts a schematic of an exemplary propionate biosynthesis gene cassette.

FIG. 20 depicts a schematic of an exemplary propionate biosynthesis gene cassette.

FIG. 21 depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production. Glucose and glycerol dissimilation pathways are shown under microaerobic conditions. In vivo, e.g., in a mammal, glycerol is not a substrate, and therefore only the glucose pathway is utilized.

FIG. 22 depicts a propionate production strategy. FIG. 22A a schematic of a construct comprising the sleeping beauty mutase operon from E. coli under the control of a heterologous FnrS promoter. FIG. 22B depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 22A.

FIG. 23 depicts a schematic of a construct comprising GLP-1 (1-37) under the control of the FliC promoter and 5′UTR containing the N-terminal flagellar secretion signal for secretion.

FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D depict schematics of the organization of exemplary GLP-1 secretion constructs with phoA (FIG. 24A and FIG. 24B) or OmpA (FIG. 24C and FIG. 24D) secretion tags. Three different RBS binding sites, 20K (FIG. 24A and FIG. 24C), 100K (FIG. 24B), and 67K (FIG. 24D) with varying strength (20<67<100) are used. In some embodiments, the Tet inducible promoter and the TetR sequence is replaced by a different inducible promoter system or a constitutive promoter in these constructs. In some embodiments, the background of the strain which contains these constructs and from which GLP-1 is secreted comprises a deletion or mutation in 1pp. FIG. 24A depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -PhoA-Glp1. FIG. 24B depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-100K RBS -PhoA-Glp1. FIG. 24C depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -OmpF-Glp1. FIG. 24D depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprisesTetR-pTet-67K RBS -OmpF-Glp1.

FIG. 25A and FIG. 25B depict schematics of the genetically engineered strains SYN2627 (comprising TetR-pTet-20K RBS -PhoA-G1p1) and SYN2643 (comprising TetR-pTet-20K RBS -PhoA-G1p1). Both strains comprise a deletion or mutation in 1pp. FIG. 25C depicts a bar graph showing the intracellular and secreted levels of GLP-1 as detected by ELISA assay for strains SYN2627 and SYN2643.

FIG. 26A and FIG. 26B depict line graphs of ELISA results. FIG. 26A depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum-starved Colo205 cells treated with supernatants from engineered bacteria comprising a PAL deletion and an integrated construct encoding hIL-22 with a phoA secretion tag. The data demonstrate that hIL-22 secreted from the engineered bacteria is functionally active. FIG. 26B depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA showing a antibody completion assay. Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody. The data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.

FIG. 27 depicts bile salt metabolism. Bile salts are synthesized from cholesterol in the liver and stored in the gallbladder. After release into the duodenum, microbial bile salt hydrolase activity in the small intestine deconjugates the glycine or taurine molecules to produce primary bile acids (also known as unconjugated bile acids). Most bile acids are reabsorbed into the enterohepatic portal system, but some enter the large intestine where they are further metabolized by microbial 7α-dehydroxylase to produce secondary bile acids. Excess bile acids are also lost in the stool (200 mg-600 mg per day).

FIG. 28 depicts the structure of bile salts and the location at which bile salt hydrolase enzymes deconjugate the bile salts. BSH activity has been detected in Lactobacillus spp, Bifidobacterium spp, Enterococcus spp, Clostridium spp, and Bacteroides spp. BSH positive bacteria are gram positive with the exception of two Bacteroides strains. BSH in has been detected in pathogenic bacteria, e.g., Listeria monocytogenes and Enterococcus feacalis. E. coli does not demonstrate BSH actvity nor contain bsh homolog in genome

FIG. 29 depicts the state of one non-limiting embodiment of the bile salt hydrolase enzyme construct under inducing conditions. Expression of the bile salt hydrolase enzyme and a bile salt transporter are both induced by the FNR promoter in the absence of oxygen. The thyA gene has been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth. The recombinant bacterial cell may further comprise an auxotrophic mutation, a type III secretion system, and/or a kill switch, as further described herein.

FIG. 30 depicts schematic of the E. coli tryptophan synthesis pathway, including genes, enzymes, and reactions involved. The seven genes, or genetic segments, seven enzymes, or enzyme domains, and seven reactions, involved in tryptophan formation are shown. Only one of the reactions is reversible. The products of four other pathways contribute carbon and/or nitrogen during tryptophan formation. Two of the tryptophan pathway enzymes often function as polypeptide complexes: anthranilate synthase, consisting of the TrpG and TrpE polypeptides, and tryptophan synthase, consisting of the TrpB and TrpA polypeptides.

FIG. 31 depicts one embodiment of the disclosure in which the E. coli TRP synthesis enzymes are expressed from a construct under the control of a tetracycline inducible system.

FIG. 32 depicts a schematic of tryptophan metabolism in humans. The abbreviations for the enzymes are as follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase; AAAD: aromatic-amino acid decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase; HIOMT, hydroxyl-O-methyltransferase; IDO, indoleamine 2,3-dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3-monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan 2,3-dioxygenase; TPH, tryptophan hydroxylase; QPRT, quinolinic acid phosphoribosyl transferase. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the tryptophan metabolism enzymes depicted in FIG. 32, or bacterial functional homologs thereof. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes which produce one or more of the tryptophan metabolites depicted in FIG. 32. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments, the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

FIG. 33 depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function. I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production. Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections. Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon-like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).

FIG. 34 depicts a schematic of the trypophan metabolic pathway. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 34. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 34, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters.

FIG. 35A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g., in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase (EC1.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2-monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines (-) indicate a spontaneous reaction. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 35. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 35. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments, the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters. FIG. 35B Depicts a schematic of tryptophan derived pathways. Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole-3-acetaldehyde; IAA: Indole-3-acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole-3-pyruvic acid; IAM: Indole-3-acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3-acetonitrile; N-formyl Kyn: N-formylkynurenine; Kyn:Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3′-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole.

FIG. 36A, FIG. 36B, FIG. 36C, and FIG. 36D depicts schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). FIG. 36A shows a schematic depicting an exemplary Tryptophan circuit. Tryptophan is produced from its precursor, chorismate, through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan repressor trpR is also depicted. Optional production of chorismate through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36C, and/or FIG. 36D. FIG. 36B depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36C, and/or FIG. 36D. Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. FIG. 36C depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene. Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD1 to NADH. E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36D. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. FIG. 36D depicts a non-limiting example of a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further optionally comprises either a wild type or a feedback resistant SerA gene. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36C. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. Optionally, the bacteria may also comprise a deletion in PheA, which prevents conversion of chorismate into phenylalanine and thereby promotes the production of anthranilate and tryptophan. FIG. 37A, FIG. 37B, FIG. 37D, FIG. 37D, FIG. 37E, FIG. 37F, FIG. 37G, and FIG. 37H depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included. FIG. 37A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. In certain embodiments the one or more cassettes are under the control of inducible promoters. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for Tryptophan decarboxylase, e.g., from Catharanthus roseus, which converts tryptophan to tryptamine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli ), which converts tryptophan to indole-3-acetaldehyde and FICZ, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), which together convert tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) which together convert tryptophan to kynurenine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g.,from homo sapiens or AADAT (Kynurenine/alpha-amino adipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens, which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter. FIG. 37G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from E. coli ), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. The genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g., from Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in any of FIG. 37A, FIG. 37B, FIG. 37D, FIG. 37D, FIG. 37E, FIG. 37F, FIG. 37G and FIG. 37H may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.

FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In certain embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. In FIG. 38A, the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana) which together produce indole-3-acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38B the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes) of tynA (Monoamine oxidase, e.g., from E. coli ) and or iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38C the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana) e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38D the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38E the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana and cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nitl (Nitrilase, e.g., from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter. the engineered bacterium shown in any of FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth. In FIG. 38F the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

FIG. 39A, FIG. 39B, and FIG. 39C depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). FIG. 39A a depicts non-limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine. FIG. 39B depicts a non-limiting example of an indole-3-acetate producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate. FIG. 39C depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises a circuit as described in FIG. 44, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol-3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH/ and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole-3-lactate).

FIG. 40A and FIG. 40B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production. FIG. 40A depicts a schematic showing intermediates in tryptophan biosynthesis and the gene products catalyzing the production of these intermediates. Phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon. Alternatively, chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the serine biosynthesis pathway, D-3-phosphoglycerate is converted to serine, which can also be a source for tryptophan biosynthesis. AroG AroF, AroH: DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). There are three isozymes of DAHP synthase, each specifically feedback regulated by tyrosine (AroF), phenylalanine (AroG) or tryptophan(AroH). AroB: Dehydroquinate synthase (DHQ synthase) is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to dehydroquinate (DHQ). AroD: 3-Dehydroquinate dehydratase (DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the conversion of DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic ring. AroE, YdiB: E. coli expresses two shikimate dehydrogenase paralogs, AroE and YdiB. Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme converts 3-dehydroshikimate to shikimate by catalyzing the NADPH linked reduction of 3-dehydro-shikimate. AroL/AroK: Shikimate kinase is involved in the fifth step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. There are two shikimate kinase enzymes, I (AroK) and II (AroL). AroA: 3-Phosphoshikimate-1-carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. EPSP synthase catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of phosphate to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP). AroC: Chorismate synthase (AroC) is involved in the 7th and last step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system. TrpEDCAB (E coli trp operon): TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L-glutamate. Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5′-phosphoribosyl)-anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional phosphoribosylanthranilate isomerase/indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway. The phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into carboxyphenylaminodeoxyribulose phosphate. The indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure of this product to yield indole-3-glycerol phosphate. The TrpA polypeptide (TSase α) functions as the a subunit of the tetrameric (α2-β2) tryptophan synthase complex. The TrpB polypeptide functions as the 0 subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the β reaction. TnaA: Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L-tryptophan to indole, pyruvate and NH4+. PheA: Bifunctional chorismate mutase/prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional chorismate mutase/prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine aminotransferase (TyrB), also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. TyrB catalyzes the transamination of 2-ketoisocaproate, p-hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and phenylalanine, respectively. TyrB overlaps with the catalytic activities of branched-chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine. SerA: D-3-phosphoglycerate dehydrogenase catalyzes the first committed step in the biosynthesis of L-serine. SerC: The serC-encoded enzyme, phosphoserine/phosphohydroxythreonine aminotransferase, functions in the biosythesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5′-phosphate is a cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated. FIG. 40B depicts a schematic showing exemplary engineering strategies which can improve tryptophan production. Each of these exemplary strategies can be used alone or two or more strategies can be combined to increase tryptophan production. Intervention points are in bold, italics and underlined. In one embodiment of the disclosure, bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEtbr, and/or TrpD, and/or TrpC, and/or TrpA, and/or TrpB. In one embodiment, endogenous TnaA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise one or more additional copies of SerA. In one embodiment, bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter. In one embodiment, endogenous PheA is knocked out through mutation(s) and/or deletion(s). In one embodiment, two or more of the strategies depicted in the schematic of FIG. 40B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.

FIG. 41A and FIG. 41B and FIG. 41C depict bar graphs showing tryptophan production by various engineered bacterial strains. FIG. 41A depicts a bar graph showing tryptophan production by various tryptophan producing strains. The data show expressing a feedback resistant form of AroG (AroG^fbr) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE^fbr) has a positive effect on tryptophan production. FIG. 41B shows tryptophan production from a strain comprising a tet-trpE^fbrDCBA, tet-aroG^fhr construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically). FIG. 41C depicts a bar graph showing improved tryptophan production by engineered strain comprising ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-aroG^fbr through the addition of serine.

FIG. 42 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476. SYN2126 ΔtrpRΔtnaA. ΔtrpRΔtnaA, tet-aroGfbr. SYN2339 comprises ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEtbrDCBA. SYN2473 comprises ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA. SYN2476 comprises ΔtrpRΔtnaA, tet-trpEtbrDCBA. Results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

FIG. 43 depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit. IPA produced by the gut microbiota has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate Gastrointestinal Bardrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

FIG. 44 depicts a schematic of indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA) circuits. Enzymes are as follows : 1. TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldH1/FldH2: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; 1ad1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.

Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and H⁺. Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3yl)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

FIG. 45 depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iad1 incorporated at the end of the second construct (AtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA.

FIG. 46 depicts a bar graph showing tryptophan and tryptamine production for strains SYN2339, SYN2340, and SYN2794. SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine and comprises ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tetaroG^fhr (p15A). SYN2340 comprises ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tetaroG^fbr-tdc_Cr (p15A). SYN2794 comprises ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tetaroG^fbr-tdc_Cs (p15A). Results indicate that Tdc_Cs from Clostridium sporogenes is more efficient the Tdc_Cr from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.

FIG. 47 depicts a schematic of an E. coli that is genetically engineered to express a butyrate cassette.

FIG. 48 depicts a schematic of an E. coli that is genetically engineered to express a a propionate biosynthestic cassette.

FIG. 49 depicts a schematic of an E. coli that is genetically engineered to express a GLP-1 and a secretion system as known in the art or described herein.

FIG. 50 depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynurenine biosynthetic cassette is expressed to produce Kynurenine. Both the transporter and Kynurenine biosynthetic cassette genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA).

FIG. 51 depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Tryptophan is synthesized and then Kynurenine is synthesized from the synthesized tryptophan or from tryptophan imported into the cell. Both the transporter and kynureninase biosynthetic genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA).

FIG. 52 depicts a schematic of an E. coli that is genetically engineered to express a butyrate and a propionate biosynthestic cassette.

FIG. 53 depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for kynurenine and/or tryptophan is also expressed or provided.

FIG. 54 depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to tryptamine and/or indole acetic acid or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. A tryptophan transporter for import of tryptophan also expressed. Export mechanism for kynurenine is also expressed or provided.

FIG. 55 depicts a schematic of an E. coli that is genetically engineered to produce butyrate, tryptophan metabolites, and tryptophan (which can be converted to bioactive tryptophan metabolites or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for tryptophan and/or tryptophan metabolites is also expressed or provided.

FIG. 56 depicts a schematic of an E. coli that is genetically engineered to produce butyrate, and propionate, kynurenine and/or other tryptophan metabolites, and GLP-1, under the control of a FNR-responsive promoter and further comprising a secretion system, e.g., for GLP-1 secretion as known in the art or described herein. Export mechanism for kynurenine/or tryptophan metabolites is also expressed or provided.

FIG. 57 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. The malE/K site is circled. In some embodiments of the disclosure, FNR-ArgAfbr is inserted at the malEK locus.

FIG. 58 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.

FIG. 59 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).

FIG. 60 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs. In some embodiments, an Glp-1 expression circuit, a butyrate production circuit, a propionate production circuit, and a tryptophan and/or indole metabolite biosynthetic cassette are inserted at four or more different chromosomal insertion sites

FIG. 61 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

FIG. 62 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

FIG. 63 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP-binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

FIG. 64 depicts a schematic of the outer and inner membranes of a gram-negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g., therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

FIG. 65 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).

FIG. 66A, FIG. 66B, and FIG. 66C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., metabolic and/or satiety effector and/or immune modulator polypeptides described herein, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, is assembled behind a fliC-5′UTR, and is driven by the native fliC and/or fliD promoter (FIG. 66A and FIG. 66B) or a tet-inducible promoter (FIG. 66C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in FIG. 66B and FIG. 66D.

FIG. 67A and FIG. 67B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., metabolic and/or satiety effector and/or immune modulator polypeptides described herein, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a tet promoter (FIG. 67A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, FIG. 67B), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters.

FIG. 68 depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P_araBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. FIG. 68A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. FIG. 68B depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit. FIG. 68C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.

FIG. 69 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

FIG. 70 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

FIG. 71 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.

FIG. 72 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

FIG. 73 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-316.

FIG. 61A, FIG. 74B, FIG. 74C, and FIG. 74D depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (FIG. 74A and FIG. 74B), which also contains a chromosomal component (shown in FIG. 74C and FIG. 74D). The Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (FIG. 74A) and thyA (FIG. 74B) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, and one or more metabolic and/or satiety effector(s) and/or immune modulator are expressed from an inducible or constitutive promoter. FIG. 74C and FIG. 74D depict schematics of the gene organization of the chromosomal component of a biosafety system. FIG. 74C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter. FIG. 74D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. If the plasmid containing the functional DapA is used (as shown in FIG. 74A), then the chromosomal constructs shown in FIG. 74C and FIG. 74D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in FIG. 74B), then the chromosomal constructs shown in FIG. 74C and FIG. 74D are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.

FIG. 75 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown in Table 2 (Pfnr1-5). Different FNR-responsive promoters were used to create a library of anaerobic-inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+O₂) or anaerobic conditions (−O₂). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.

FIG. 76A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P_fnrs). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. FIG. 76B depicts FNR promoter activity as a function of (3-galactosidase activity in SYN340. SYN340, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr under anaerobic conditions. FIG. 76C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

FIG. 77A and FIG. 77B depict schematics of ATC (FIG. 77A) or nitric oxide-inducible (FIG. 77B) reporter constructs. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible P_tet-GFP reporter construct or the nitric oxide inducible P_nsrR-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. FIG. 77C depicts a schematic of the constructs. FIG. 77D depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter. DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.

FIG. 78A depicts a “Oxygen bypass switch” useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more metabolic and/or satiety effector(s) (POI1) and /or immune modulator and/or one or more transporter(s)/importer(s) and/or exporter(s) (PGI2) under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active payload(s) prior to administration. This can be done by pre-inducing the expression of these enzymes as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites.

FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J, The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.

In some embodiments, a Lad promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

FIG. 78B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of FnrS24Y expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bioinformatics tools for optimization of RBS are known in the art.

FIG. 78C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bioinformatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of PfnrS-POI constructs are maintained to allow for strong in vivo induction.

FIG. 79 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.

FIG. 80 depicts the gene organization of an exemplary construct, comprising a cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.

FIG. 81 depicts the gene organization of an exemplary construct comprising Lad in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI, e.g., one or more metabolic effector(s) described herein). In some embodiments, this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.

FIG. 82A, FIG. 82B, and FIG. 82C depict schematics of non-limiting examples of constructs expressing a protein of interest (POI). FIG. 82A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C. but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, the construct comprises SEQ ID NO: 101. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).

FIG. 82B depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a bio safety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

FIG. 82C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter. The arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POI(s) prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a bio safety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

FIG. 83A depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta-galactosidase activity was measured. FIG. 83B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA—as the case may be—are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).

FIG. 84 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

FIG. 85 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating. FIG. 85 depicts a bar graph of residence over time for streptomycin resistant Nissle.

FIG. 86 depicts a schematic diagram of a wild-type clbA construct (upper panel) and a schematic diagram of a clbA knockout construct (lower panel).

FIG. 87 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animla disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.

FIG. 88A, B, C, D, and E depict a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. FIG. 88A depicts the parameters for starter culture 1 (SC1): loop full—glycerol stock, duration overnight, temperature 37° C., shaking at 250 rpm. FIG. 88B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C., shaking at 250 rpm. FIG. 88C depicts the parameters for the production bioreactor: inoculum—SC2, temperature 37° C., pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. FIG. 88D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1×10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. FIG. 88E depicts the parameters for vial fill/storage: 1-2 mL aliquots, −80° C.

DESCRIPTION OF EMBODIMENTS

The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating metabolic diseases. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s), or a gene cassette(s) encoding one or more non-native biosynthetic pathway(s) for producing one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s), or a gene cassette(s) encoding one or more non-native biosynthetic pathway(s) for producing one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s) and further comprise gene sequence encoding one or more immune modulator molecules, such as any of the immune modulators described herein. In some embodiments, the gene sequence or gene cassette is further operably linked to an inducible promoter, for example, a regulatory region that is controlled by a transcription factor that is capable of sensing low-oxygen conditions, inflammatory conditions, or other tissue-specific or environment-specific conditions. In certain embodiments, the genetically engineered bacteria are capable of producing metabolic and/or satiety effector molecule and/or anti-inflammatory molecules in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used in order to treat and/or prevent conditions associated with metabolic diseases, including obesity and type 2 diabetes.

In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

As used herein, “metabolic diseases” include, but are not limited to, type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.

Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility.

Metabolic diseases are associated with a variety of physiological changes, including but not limited to elevated glucose levels, elevated triglyceride levels, elevated cholesterol levels, insulin resistance, high blood pressure, hypogonadism, subfertility, infertility, abdominal obesity, pro-thrombotic conditions, and pro-inflammatory conditions. A metabolic effector is a molecule that is capable of minimizing any one or more of said physiological changes. For example, a metabolic effector molecule may enhance the body's sensitivity to insulin, thereby ameliorating insulin resistance. Insulin resistance is a physiological condition in which the body's insulin becomes less effective at lowering blood sugar. Excess blood sugar can cause adverse health effects such as type 2 diabetes. “Satiety” is used to refer to a homeostatic state in which a subject feels that hunger or food craving is minimized or satisfied. A satiety effector is a molecule that contributes to the minimization or satisfaction of said hunger or food craving. A molecule may be primarily a metabolic effector or primarily a satiety effector. A molecule may be both a metabolic and satiety effector, e.g., GLP-1.

“Metabolic effector molecules” and/or “satiety effector molecules” include, but are not limited to, n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). Such molecules may also include compounds that inhibit a molecule that promotes metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor. A metabolic and/or satiety effector molecule may be encoded by a single gene, e.g., glucogon-like peptide 1 is encoded by the GLP-1 gene. Alternatively, a metabolic and/or satiety effector molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., propionate. These molecules may also be referred to as therapeutic molecules.

An “anti-inflammatory” or anti-inflammatory molecule” refers to a molecule that reduces, decreases, inhibits, or prevents an inflammatory response, either directly or indirectly. Non-limiting examples of anti-inflammatory molecules include short-chain fatty acids (e.g., butyrate, propionate, acetate), certain tryptophan metabolites, e.g., indoles and indole metabolites, as described herein, certain cytokines, including but not limited to, IL-10, IL-22, IL-4, IL-13, IFNa, and TGFB.

An “immune modulator” or “immune modulator molecule” refers to a molecule that modulates an inflammatory response. Non-limiting examples of immune modulator molecules include molecules that directly modulate an inflammatory response and also includes molecules that activate (stimulate or increase the activity of) or inhibit (decrease the activity of) molecules that directly modulate an inflammatory response. For example, an immune modulator can decrease levels of inflammatory growth factors and cytokines, e.g., IL-1β, IL-6, and/or TNF-α and proinflammatory signaling, e.g. NF-kappaB signaling and/or can increase levels of anti- inflammatory growth factors and cytokines, e.g., IL4, IL-10, IL-13, IFN-alpha and/or transforming growth factor-beta. Other immune modulators include, but are not limited to, short-chain fatty acids (e.g., butyrate, propionate, acetate), certain tryptophan metabolites, e.g., indoles and indole metabolites, as described herein, certain cytokines, including but not limited to, IL-10, IL-22, IL-4, IL-13, IFNa, and TGFB.

As used herein, the term “engineered bacterial cell” or “engineered bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

A “programmed bacterial cell” or “programmed engineered bacterial cell” is an engineered bacterial cell that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

As used herein, a “heterologous” gene or “heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

As used herein, the term “coding region” refers to a nucleotide sequence that codes for a specific amino acid sequence. The term “regulatory sequence” refers to a nucleotide sequence located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.

As used herein, a “gene cassette” or “operon” or “genetic circuit” encoding a biosynthetic pathway or catabolic pathway refers to the two or more genes that are required to produce a metabolic and/or satiety effector and/or immune modulator molecule, e.g., propionate and/or immune modulator molecule (e.g., tryptophane metabolite, e.g., indole). In addition to encoding a set of genes capable of producing said molecule, the gene cassette or operon or “genetic circuit” may also comprise additional transcription and translation elements, e.g., a ribosome binding site.

A “butyrogenic gene cassette,” “butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. The butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB.

Likewise, a “propionate gene cassette” or “propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway. Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyses the reduction of lactate to propionate. Dehydration of (R)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the acuI gene from R. sphaeroides. This gene product catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of Acul in E coli, YhdH is used (see.e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004). This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.

In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-methylmalonylCoA, YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG (methylmalonylCoA decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH (propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009). This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD 18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.

The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

An “acetate gene cassette” or “acetate operon” refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria “synthesize acetate from a number of carbon and energy sources,” including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria. Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or CO₂+H₂ into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.

Each gene or gene cassette may be operably linked to a promoter that is induced under low-oxygen conditions. “Operably linked” refers a nucleic acid sequence, e.g., a gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate, and/or immune modulator. In the presence of an inducer of said regulatory region, a metabolic and/or satiety effector and/or immune modulator molecule is expressed. An “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a gene encoding a first molecule, e.g., a transcription factor, which is capable of regulating a second regulatory region that is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate, and/or immune modulator. In the presence of an inducer of the first regulatory region, the second regulatory region may be activated or repressed, thereby activating or repressing production of propionate. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.”

“Exogenous environmental condition(s)” or “environmental conditions” refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment. Thus, “exogenous” and “endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease-state, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.

As used herein, “exogenous environmental conditions” or “environmental conditions” also refers to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the microorganism. “Exogenous environmental conditions” may also refer to the conditions during in vitro growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations. Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium. Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions. Such conditions affect proliferation rate, rate of induction of the protein of interest and overall viability and metabolic activity of the strain during strain production.

In some embodiments, the gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule is operably linked to an oxygen level-dependent regulatory region such that the effector molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, the oxygen level-dependent regulatory region is operably linked to a propionate gene cassette; in low oxygen conditions, the oxygen level-dependent regulatory region is activated by a corresponding oxygen level-sensing transcription factor, thereby driving expression of the propionate gene cassette. Examples of oxygen level-dependent transcription factors and corresponding promoters and/or regulatory regions include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.

TABLE 1
Examples of transcription factors and
responsive genes and regulatory regions
Transcription Examples of responsive genes,
Factor        promoters, and/or regulatory regions:
FNR           nirB, ydfZ, pdhR, focA, ndH, hlyE, narK,
              narX, narG, yfiD, tdcD
              Table 4
ANR           arcDABC
DNR           norb, norC

As used herein, a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR-responsive promoter operably linked to a propionate gene cassette.

“Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli σ^S promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ³² promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ⁷⁰ promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis σ^A promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P_liaG (BBa_K823000), P_lepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis σ^B promoter (e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)).

“Gut” refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

“Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules. In certain aspects, the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.

“Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Pat. No. 6,835,376; U.S. Pat. No. 6,203,797; U.S. Pat. No. 5,589,168; U.S. Pat. No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

“Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. No. 5,589,168; U.S. Pat. No. 6,203,797; U.S. Pat. No. 6,835,376). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

As used herein, the terms “secretion system” or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g.,HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a “secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the “passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu. In some embodiments, the secretion system involves the generation of a “leaky” or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from 1pp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

As used herein, the term “modulate” and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement. Thus, for example, “modulate” or “modulation” includes up-regulation and down-regulation. A non-limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound). Another non-limiting example of modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen. In another non-limiting example, modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms. Thus, “modulate” is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms).

As used herein, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a propionate gene cassette, which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and/or propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically modified bacterium comprising a propionate gene cassette, in which the plasmid or chromosome carrying the propionate gene cassette is stably maintained in the host cell, such that the gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo.

As used herein, the term “treat” and its cognates refer to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, “treat” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “treat” refers to inhibiting the progression of a disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “treat” refers to slowing the progression or reversing the progression of a disease or disorder. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease or disorder.

Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treating metabolic diseases may encompass reducing or eliminating associated symptoms, e.g., weight gain, and does not necessarily encompass the elimination of the underlying disease or disorder, e.g., congenital leptin deficiency. Treating the diseases described herein may encompass increasing levels of propionate, increasing levels of butyrate, and increasing GLP-1, and/or modulating levels of tryptophan and/or its metabolites (e.g., kynurenine), and does not necessarily encompass the elimination of the underlying disease.

As used herein a “pharmaceutical composition” refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and/or excipient.

The phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.

The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., obesity. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a metabolic disease. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

The phrase “and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list.

Bacteria

The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a non-native metabolic and/or satiety effector and/or immune modulator molecule, wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterobacteriaceae family that “has evolved into one of the best characterized probiotics” (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli α-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and is not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle's “therapeutic efficacy and safety have convincingly been proven” (Ukena et al., 2007). In a recent study in non-human primates, Nissle was well tolerated by female cynomolgus monkeys after 28 days of daily NG dose administration at doses up to 1×1012 CFU/animal. No Nissle related mortality occurred and no Nissle related effects were identified upon clinical observation, body weight, and clinical pathology assessment (see, e.g., PCT/US16/34200).

One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. It is known, for example, that “the clostridial butyrogenic pathway [genes] . . . are widespread in the genome-sequenced clostridia and related species” (Aboulnaga et al., 2013). Furthermore, genes from one or more different species of bacteria can be introduced into one another, e.g., the butyrogenic genes from Peptoclostridium difficile have been expressed in Escherichia coli (Aboulnaga et al., 2013).

Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009). Thus the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria.

In certain embodiments, the payload(s) described below are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the payload is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.

Metabolic Diseases

NASH

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), where excess fat accumulation in the liver results in chronic inflammation and damage. Nonalcoholic fatty liver disease is a component of metabolic syndrome and a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Simple liver steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH, which tends to progress to cirrhosis in up to 20% of patients and can subsequently lead to liver failure or hepatocellular carcinoma. NASH affects approximately 3-5% of the population in America, especially in those identified as obese. NASH is characterized by such abnormalities as advanced lipotoxic metabolites, pro-inflammatory substrate, fibrosis, and increased hepatic lipid deposition. If left untreated, NASH can lead to cirrhosis, liver failure, and hepatocellular carcinoma.

Although patients diagnosed with alcoholic steatohepatitis demonstrate similar symptoms and liver damage, NASH develops in individuals who do not consume alcohol, and the underlying causes of NASH are unknown. Hepatic steatosis occurs when the amount of imported and synthesized lipids exceeds the export or catabolism in hepatocytes. An excess intake of fat or carbohydrate is the main cause of hepatic steatosis. NAFLD patients exhibit signs of liver inflammation and increased hepatic lipid accumulation. In addition, the development of NAFLD in obese individuals is closely associated with insulin resistance and other metabolic disorders and thus might be of clinical relevance). Therfore, Possible causative factors include insulin resistance, cytokine imbalance (specifically, an increase in the tumor necrosis factor-alpha (TNF-α)/adiponectin ratio), and oxidative stress resulting from mitochondrial abnormalities.

Currently, there is no accepted approach to treating NASH. Therapy generally involves treating known risk factors such as correction of obesity through diet and exercise, treating hyperglycemia through diet and insulin, avoiding alcohol consumption, and avoiding unnecessary medication. In animal models, administration of butyrate has been shown to reduce hepatic steatosis, inflammation, and fat deposition (see, for example, Jin et al., British J. Nutrition, 114(11):1745-1755, 2015 and Endo et al., PLoS One, 8(5):e63388, 2013). Colonic propionate delivery has also been shown to reduce intrahepatocellular lipid content in NASH patients, including improvements in weight gain and intra-abdominal fat deposition (see, for example, Chambers et al., Gut, gutjnl-2014), and GLP-1 administration has been shown to reduce the degree of lipotoxic metabolites and pro-inflammatory substrates, both of which have been shown to speed NASH development, as well as reduce hepatic lipid deposition (see, for example, Bernsmeier et al., PLoS One, 9(1):e87488, 2014 and Armstrong et al., J. Hepatol., 2015).

The liver has both an arterial and venous blood supply, with the majority of hepatic blood flow coming from the gut via the portal vein. In NASH the liver is exposed to potentially harmful substances derived from the gut (increased perability and reduced intestinal integrity), including translocated bacteria, LPS and endotoxins as well as secreted cytokines. Translocated microbial products might contribute to the pathogenesis of fatty liver disease by several mechanisms, including stimulating pro-inflammatory and profibrotic pathways via a range of cytokines. For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity.

The role of bile acids in the pathogenesis of NAFLD and NASH has been extensively studied (Leung et al., The Role Of The Gut Microbiota In NAFLD; Nature Reviews I Gastroenterology & Hepatology). For example, in one study study, manipulation of the gut microbiota changed intestinal bile acid composition leading to intestinal antagonism of FRX, the master regulator of bile acid metabolism. This FXR antagonism reduced ceramide synthesis and de novo lipogenesis in the liver (Jiang, C. et al. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. J. Clin. Invest. 125, 386-402 (2015)).

Studies have also suggested that rapid weight loss through bariatric surgery (e.g. gastric bypass) is effective in decreasing steatosis, hepatic inflammation, and fibrosis. Other treatments have involved using anti-diabetic medications such as metformin, rosiglitazone, and pioglitazone. Though inconclusive, the studies suggest that the medications stimulate insulin sensitivity in NASH patients, thus alleviating liver damage. In cases were NASH has resulted in advanced cirrhosis, the only treatment is a liver transplant. Regardless, no current treatments are wholly determinative or reliable for treating NASH. Therefore, a need exists for improved therapies and treatments of NASH.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate and further comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32.

In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein., e.g., for the treatment, prevention and/or management of NASH

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in

FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios, e.g., for the prevention, management and/or treatment of NASH.

In certain embodiments, one or more of these circuits may be combined for the treatment of NASH and/or NAFLD. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of NASH and/or NAFLD.

Diabetes

Diabetes mellitus type 1 (also known as type 1 diabetes) is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. The subsequent lack of insulin leads to increased glucose in blood and urine. The classical symptoms are frequent urination, increased thirst, increased hunger, and weight loss. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of diabetes mellitus.

Diabetes mellitus type 2 is a long term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin. Common symptoms include increased thirst, frequent urination, and unexplained weight loss. Symptoms may also include increased hunger, feeling tired, and sores that do not heal. Often symptoms come on slowly. Long-term complications from high blood sugar include heart disease, strokes, diabetic retinopathy which can result in blindness, kidney failure, and poor blood flow in the limbs which may lead to amputations.

Insulin resistance (IR) is generally regarded as a pathological condition in which cells fail to respond to the normal actions of the hormone insulin. Normally insulin produced when glucose enters the circulation after a meal triggers glucose uptake into cells. Under conditions of insulin resistance, the cells in the body are resistant to the insulin produced after a meal, preventing glucose uptake and leading to high blood sugar.

The kynurenine hypothesis of diabetes is based on evidence of diabetogenic effects of the kynurenine metabolite Xanthurenic Acid (XA) and the realization that the KP is upregulated by low-grade inflammation and stress, two conditions involved in the pathogenesis of insulin resistance, and of diabetes type I and diabetes type II. Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, possibly due to chronic stress or the low-grade inflammation, which are risk factors for T2DM. As such, the production of kynurenine metabolites can function as a regulatory mechanism to attenuate damage by the inflammation-induced production of reactive oxygen species.

Experimental and clinical data have clearly established that besides fat, muscle and liver, pancreatic islet tissue itself is a site of inflammation during obesity and type 2 diabetes. It is therefore conceivable that in parallel to the high free fatty acids and glucose levels, pancreatic islet exposure to increased levels of cytokines may induce dysregulation of islet KP.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of type 2 diabetes (T2DM). In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein., in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In one embodiment, the genetically engineered bacteria produce IL-22, e.g., for the treatment of diabetes and other metabolic disease described herein.

In certain embodiments, one or more of these circuits may be combined for the treatment of type 2 diabetes. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of type 2 diabetes.

Obesity

Metabolic Syndrome affects approximately 20-30% of the middle-aged population, and represents an increased risk to cardiovascular disorders, the leading cause of death in the United States. Obesity, dyslipidemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of metabolic syndrome and /or obesity. Several of the metabolites and polypeptides produced by the genetically engineered bacteria are useful for increasing insulin secretion and promoting satiety, e.g. GLP-1.

Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation. Endotoxemia, a process resulting from translocation of endotoxic compounds (lipopolysaccharides [LPS]), of gram-negative intestinal bacteria. In the last decade, it has become evident that insulin resistance and T2DM are characterized by low-grade inflammation. In this respect, LPS trigger a low-grade inflammatory response, and the process of endotoxemia can therefore result in the development of insulin resistance and other metabolic disorders. Several of the metabolites produced by the genetically engineered bacteria described herein are useful in the reduction of inflammation. For example, butyrate, contributes to maintaining intestinal integrity. Other anti-inflammatory metabolites as described herein may also be useful in the treatment of type 2 diaberes.

Over nutrition leads to an excess intake of tryptophan (TRP)—an essential amino acid, a precursor for serotonin (5-HT) and melatonin, and a key player in the caloric intake regulation. Yet, the circulating levels of TRP have been shown to be low in morbidly obese subjects (Brandacher G, Winkler C, Aigner F, et al. Bariatric surgery cannot prevent tryptophan depletion due to chronic immune activation in morbidly obese patients. Obes Surg 2006; 16:541-548).

Serotonin regulates carbohydrate and fat intake (Blundell J E, Lawton C L. Serotonin and dietary fat intake: effects of dexfenfluramine. Metabolism 1995; 44:33-37) , relieves stress which is another caloric intake trigger (Buwalda B, Blom W A, Koolhaas J M, van Dijk G. Behavioral and physiological responses to stress are affected by high-fat feeding in male rats; Physiol Behav 2001; 73:371-377), and inhibits neuropeptide Y (NYP)—one of the most potent orexigenic peptides in the hypothalamus (Jia Y, El-Haddad M, Gendy A, Nguyen T, Ross M G.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1 and/or GLP-1 analog(s). In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of obesity. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including, but not limited, to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, and elsewhere herein, including but not limited to, Tryptamine, Indole-3-acetaldehyde, Indole-3-acetic acid, indole-3- propionic acid, Indole, 6-formylindolo(3,2-b)carbazole, Kynurenic acid, Indole-3-aldehyde; 3,3′ -Diindo lylmethane. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of obesity. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of obesity. Further combinations may include cytokine producing circuits, such as IL-22.

Prader Willi Syndrome

Prader-Willi syndrome (OMIM 176270) is a complex genetic neurodevelopmental disorder with manifested early in failure to thrive, feeding difficulties during infancy, hypogonadism/hypogenitalism, growth hormone deficiency, and typically a paternal 15q11-q13 chromosome deletion. In early childhood trough alduhood, food seeking behaviors and hyperphagia are noted along with a low metabolic rate and decreased physical activity leading to obesity which can be life-threatening, if not controlled. PWS is considered the most common syndromic cause of life threatening obesity in childhood (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). It has been reported that, when matched for body mass index (BMI), PWS adults had the same prevalence of metabolic syndrome (41.4%) and insulin resistance index as obese controls.

Prader-Willi syndrome (PWS) has no cure. PWS syndrome individuals present with obesity with hyperphagia and deficit of satiety, and in some cases insulin resistance, that persists thoughout youth and adulthood and remains a critical problem in PWS teenagers and adults because it leads to severe complications, such as limb edema, cardiac or respiratory failure, and physical disabilities. Severe obesity, and food seeking therfroe remains the larges problem with PWS. Access to food must be strictly supervised and limited. Therefore, agents which modulate satiety and orh insulin levels may be useful in the treatment of PWS.

In additiona, increased inflammatory markers and cytokine levels in the plasma have been observed in PWS individuals. These cytokines serve as chemoattractants for recruitment of immune cells and indicate an inflammatory component in PWS, which underlies certain aspects of the pathology (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). Therefore, anti-inflammatory agents may be useful in the treatment of certain aspects of PWS.

In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of PWS. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of PWS.

Metabolic Syndrome

Metabolic syndrome is a clustering of at least three of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of metabolic syndrome. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of metabolic syndrome.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of metabolic syndrome. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of metabolic syndrome.

Cardiovascular Disease

Metabolic syndrome is an important risk factor for cardiovascular disease incidence and mortality, as well as all-cause mortality.

Cardiovascular disease includes coronary artery diseases (CAD) such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis, caused inter alia by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, and the like.

The detection, prevention, and treatment of the underlying risk factors of the metabolic syndrome are a critical approach to lower the cardiovascular disease incidence in the general population.

Cellular adhesion molecules and oxidative stress play a role in the pathogenesis of atherosclerosis in patients with chronic kidney disease (CKD) and uremia. Uremia is condition that occurs when the kidneys no longer filter properly, and is likely to occur s in the final stage of chronic kidney disease. Several studies in CKD patients have shown that tryptophan metabolites along the kynurenine pathway are increased, possibly as consequence of inflammation. Therefore, anti-inflammatory agents may be useful in the treatment of cardiovascular disease, including CKD and artherosclerosis. In some embodiments, the genetically engineered bacteria modulate the levels of one or more of tryptophan, kynurenine, kynurenine downstream metabolites, and other tryptophan metabolites and /or modulate one or more metabolite ratios.

Ischemic stroke, which results from cerebral arterial occlusion, is becoming a major cause of morbidity and mortality in today's society and affects millions of people every year. Currently, the only approved treatment for the acute phase of stroke is the recombinant thrombolytic tissue-type plasminogen activator. Identifying molecules that contribute to the ischemic damage may help to elucidate potential therapeutic targets. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of ischemia and stroke. Inflammation and oxidative stress are also involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis, ischemic stroke, and/or chronic kidney disease. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis,.ischemic stroke, and/or chronic kidney disease.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of cardionvascular disorders. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of cardionvascular disorders.

Metabolic and Satiety Effector Molecules, and Modulators of Inflammation

The genetically engineered bacteria comprise a gene encoding a non-native metabolic and/or satiety effector and/or immune modulator molecule, and/or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, the metabolic and/or satiety effector molecule is selected from the group consisting of n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). A molecule may be primarily a metabolic effector, or primarily a satiety effector. Alternatively, a molecule may be both a metabolic and satiety effector.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) which are capable of producing an effector, which can modulate the inflammatory status. Non-limiting examples include short chain fatty acids, and tryptophan and its metabolites, including indoles, as described herein.

In some embodiments, the genetically engineered bacteria comprise a gene encoding a non-native metabolic and/or satiety effector and/or immune modulator molecule, and/or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector and/or immune modulator molecule, and further comprise gene sequence(s) and/or gene cassette(s) which are capable of producing one or more immune modulators or effector molecules which can modulate the inflammatory status, including, for example, short chain fatty acids, and tryptophan and its metabolites, including indoles, as described herein.

The effect of the genetically engineered bacteria on the inflammatory status can be measured by methods known in the art, e.g., plasma can be drawn before and after administraton of the genetically engineered bacteria. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and plasma viscosity (PV) blood tests are commonly used to detect this increase n inflammation. In some embodiments the genetically engineered bacteria modulate, e.g. decrease or increase, levels of inflammatory markers, eg.. C-reactive protein (CRP).

In some embodiments, the genetically engineered bacteria modulate, e.g. decrease, levels of inflammatory growth factors and cytokines, e.g., IL-1(3, IL-6, and/or TNF-α and proinflammatory signaling, e.g. NF-kappaB signaling. In some embodiments the genetically engineered bacteria modulate, e.g. increase, levels of anti-inflammatory growth factors and cytokines, e.g., IL4, IL-10, IL-13, IFN-alpha and/or transforming growth factor-beta.

In some embodiments, the genetically engineered bacteria produce effectors, which bind to and stimulate the aromatic hydrocarbon receptor. In some embodiments the genetically engineered bacteria stimulate AHR signaling in immune cell types, including T cells, B cells, NK cells, macrophages, and dendritic cells (DCs), and/or in epithelial cells. In some embodiments, the genetically engineered bacteria modulate, e.g., increase the levels of IL-22, e.g., through stimulation of AHR.

In some emobodiments, the genetically engineered bacteria may reduce gut permeability. In some embodiments, the genetically engineered bacteria may reduce the amounts of LPS and in the circulation, which are increase in metabolic disease, e.g., in NASH.

The gene or gene cassette for producing the metabolic and/or satiety effector molecule and/or modulator of inflammation may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions, molecules, or metabolites specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.

The gene or gene cassette for producing the metabolic and/or satiety effector and/or modulator of inflammation may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing expression of the metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the metabolic and/or satiety effector molecule. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the propionate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g. FIG. 57). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. In some embodiments, the genetically engineered bacteria of the invention are capable of expressing a metabolic and/or satiety effector and/or immune modulator molecule that is encoded by a single gene, e.g., the molecule is GLP-1 and encoded by the GLP-1 gene.

One of skill in the art would appreciate that additional genes and gene cassettes capable of producing metabolic and/or satiety effector molecules and/or modulator of inflammation are known in the art and may be expressed by the genetically engineered bacteria of the invention. In some embodiments, the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g., a ribosome binding site, to enhance expression of the therapeutic molecule.

In some embodiments, the genetically engineered bacteria produce two or more metabolic and/or satiety effector molecules and/or modulator of inflammation. In certain embodiments, the two or more molecules behave synergistically to ameliorate metabolic disease. In some embodiments, the genetically engineered bacteria express at least one metabolic effector molecule and at least one satiety effector molecule and at least one modulator of inflammation.

Short Chain Fatty Acids

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. In rodent models of genetic or diet-induced obesity, supplementation of butyrate in diet, and oral administration of acetate was shown to suppress weight gain independent of food intake suppression; Propionate was reported to inhibit food intake in humans (see, e.g., Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, and refernces therein). Therefore, the production of SCFAs is likely efficacious in the treatment of metabolic syndrome and related disorders, and/or diabetes type2, and/or obesity.

SCFAs represent a major constituent of the luminal contents of the colon. Among SCFAs butyrate is believed to play an important role for epithelial homeostasis. Acetate and propionate have anti-inflammatory properties, which are comparable to those of butyrate (Tedelind et al., World J Gastroenterol. 2007 May 28; 13(20): 2826-2832. Acetate and propionate, similar to butyrate, inhibit TNFa-mediated activation of the NF-κB pathway. These findings suggest that propionate and acetate, in addition to butyrate, could be efficacious in the treatment of inflammatory conditions.

Propionate

In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing a metabolic and/or satiety effector molecule, e.g., propionate that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.

In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 2). Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of Acul in E coli, yhdH is used. This propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAf^fbr, thrB, thrC, ilvA^fbr, aceE, aceF, and lpd, and optionally further comprise tesB. In another embodiment, the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm converts succinyl CoA to L-methylmalonylCoA, ygfG converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA into propionate and succinate into succinylCoA.

This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.

The genes may be codon-optimized, and translational and transcriptional elements may be added. Table 2-4 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 5 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.

TABLE 2
Propionate Cassette Sequences (Acrylate Pathway)
Gene sequence Description
pct           ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACT
SEQ ID NO: 1  GATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGTA
              ACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTC
              CTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGG
              CAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGC
              ATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACC
              GTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGC
              CTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATA
              TTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGC
              ACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATAT
              TACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGG
              AATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATT
              CGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAA
              AGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGA
              AGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC
              AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTAT
              CTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAAC
              AGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCAC
              CGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAA
              GAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACG
              TTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCC
              GTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGC
              GGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCT
              TTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTAC
              CAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACCT
              GGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCC
              GCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATT
              ACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGC
              CGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCG
              TTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAA
              ATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGT
              TACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACG
              GTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGACC
              CAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGA
              CGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAG
              AAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA
lcdA          ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATA
SEQ ID NO: 2  CTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCC
              GCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGGAA
              TTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTCA
              TGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGG
              AAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTAC
              GGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGC
              CATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTG
              CTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATC
              TGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT
              CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACCA
              TGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGTTC
              CGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTT
              CGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTA
              GCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATACAAG
              CCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTT
              AATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTTA
              AAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATC
              TACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGG
              TATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTATGA
              AGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTT
              TGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTAT
              GGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGCAGA
              ACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTG
              GATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAG
              TTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACG
              GTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGC
              GTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGT
              TGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA
lcdB          ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC
SEQ ID NO: 3  CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAG
              GTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAA
              ATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC
              CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTT
              TTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCGAG
              GGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTG
              CGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCC
              CAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAGCG
              GCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACA
              ACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGG
              AAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT
              GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGC
              AGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCTTA
              AGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATT
              AAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGT
              GACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATCTTTA
              ATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAA
              AGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACC
              GCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCT
              CGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATT
              AACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAAT
              GATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGT
              ACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAG
              GTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCT
              GCAGTCATTCGTCGAAATGCTTTAA
lcdC          ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC
SEQ ID NO: 4  GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG
              TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC
              AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA
              CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG
              ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT
              TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA
              AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGC
              AATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTC
              CTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAAT
              GGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAA
              GCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTG
              AGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCT
              GAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTT
              TGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCA
              GGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTAT
              CGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAGCGC
              TGTATGCTTATGAGGCCGCCCAGAAGAAGTA
etfA          ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTG
SEQ ID NO: 5  GGTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATT
              TCGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA
              AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAAT
              CGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGT
              GCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCC
              AAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGTAAT
              TTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGCGTT
              GTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCAC
              CTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCACCAG
              TAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAGATA
              CAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTGATG
              GCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGT
              GCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGG
              CGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTCGGAT
              GAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAAACGAA
              AAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTG
              GTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCACTGGCT
              GAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGCGGCTC
              GCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGG
              TTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCG
              CTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAAGA
              CTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTA
              TCTTCGACTGCGCCGATTATGGCATCACCGGTGATTTATTTAAA
              ATCGTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAACGC
              ATGA
acrB          ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG
SEQ ID NO: 6  CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA
              TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG
              GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCT
              TACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTA
              TTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGAA
              TTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGC
              GGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTTT
              GCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCCT
              CAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCGC
              AGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGT
              TGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCTG
              ATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGAC
              TCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC
              TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT
              ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT
              TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG
              ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAAA
              CATGTCATCTAA
acrC          ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGGA
SEQ ID NO: 7  CTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGC
              GTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGATT
              GATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGC
              GGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGGTG
              GCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAGAA
              TTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATTCA
              TCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGAAC
              AGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAACTG
              GGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGG
              CGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAACGAGT
              ACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCGGGCGC
              GCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGG
              TCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCGG
              GCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGATCGCAGGT
              TCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGC
              TGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTGCTA
              TGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGCA
              ATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGT
              TCACGAGCGCATTCAATTTGGTAAACCGATCGCGAATCTGCAGG
              GAATTCAATGGTATATCGCGGATATGGCGACCAAAACCGCCGCG
              GCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGG
              TAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCTGAACGCCT
              CCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCAC
              GGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTA
              TCGCGATGCTAAGATTACGGAAATTTACGAGGGGACATCAGAAA
              TCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA
thrfbr        ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAGA
SEQ ID NO: 8  ACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGGC
              AGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCACC
              AACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAGGA
              TGCTTTACCCAATATCAGCGATGCCGAACGTATTTTTGCCGAAC
              TTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGGCG
              CAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAACA
              TGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAGCA
              TCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCGCC
              ATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACTGT
              TATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCTCG
              AATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGGCA
              AGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTCAC
              CGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCAACG
              GTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGCGCC
              GATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATACCTG
              CGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGATGT
              CCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAAGTT
              CTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATCCC
              TTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTACGC
              TCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGGGC
              ATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGTCC
              GGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTGCAG
              CGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAATCA
              TCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGACTG
              TGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTGGAAC
              TGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAACGGCTG
              GCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTGCGTGG
              GATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAATATCA
              ACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAATCTCT
              GTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGCGTTAC
              TCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGTGTTTG
              TGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGCAACTG
              AAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGACTTACG
              TGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCAATGTAC
              ATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGCGCAAGCC
              AAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCGTGAAAGA
              ATATCATCTGCTGAACCCGGTCATTGTTGACTGCACTTCCAGCC
              AGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGAAGGTTTC
              CACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGTCGATGGA
              TTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCGCGGCGTA
              AATTCCTCTATGACACCAACGTTGGGGCTGGATTACCGGTTATT
              GAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAATTGATGAA
              GTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTTCGGCA
              AGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACGCTGGCG
              CGGGAAATGGGTTATACCGAACCGGACCCGCGAGATGATCTTTC
              TGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTCGTGAAA
              CGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGAACCTGTG
              CTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCGCTTTTAT
              GGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGCGCGTGG
              CGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTTGGCAAT
              ATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGAAGTGGA
              TGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAAAACGCCC
              TGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTTGGTACTG
              CGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCGGTGTCTT
              TGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGAGTCTGA
thrB          ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT
SEQ ID NO: 9  CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG
              CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC
              AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA
              ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC
              AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAAG
              AATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGGT
              GGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCCGC
              TTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAGGC
              CGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGTTT
              TCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCATCA
              GCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGGCG
              TATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATTTT
              ACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCGAC
              ATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTGAG
              CTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTACCG
              TGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGGTCG
              CGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGCCCG
              ACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCGCGT
              TGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAAGGTT
              TTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGTACTG
              GAAAACTAA
thrC          ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT
SEQ ID NO: 10 TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTGT
              TTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGAT
              GAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCCT
              CTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAAG
              AGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAAT
              GTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAAC
              GCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATGC
              TGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGCG
              ACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACGG
              TTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAAA
              TCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAAT
              ATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGGC
              GCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGCGC
              TAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGCTG
              GCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCGCA
              GGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAAACT
              TCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGTCTG
              CCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATACCGT
              GCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGACTC
              AGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAACAAC
              TGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGCAACT
              GAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCACGCAAC
              AGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCGGAGCCG
              CACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTTGAATCC
              AGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGGCGAAAT
              TTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTTGGATCTG
              CCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGCTTTCACA
              TAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGATGATGA
              ATCATCAGTAA
ilvAfbr       ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGGC
SEQ ID NO: 11 TAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGCGC
              AGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGTAT
              TGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTTAA
              GCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGGTG
              CGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGATGCA
              GGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGTGGC
              CTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATGTTC
              CTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTTCAC
              GGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTTCGA
              CGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACCGGCG
              CAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCATCGGT
              CAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTTCCAT
              GGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGTGGCG
              GACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGCACCT
              CGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCATCCAT
              GCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAGACTG
              TTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGGAGAT
              CTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTGCACAT
              GATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCTCGATC
              TTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCGCGCTG
              TCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGTTCTGC
              AGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCTGCGTT
              ATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTTTGAAG
              CACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAGTTGCG
              TCACTTCCTGGAAGATATCCTGGGACCGGATGATGACATCACGC
              TGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTACTGCG
              TTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGATTCTTT
              GCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCGCCTCG
              AGCCGGGCACCCCTGAGTACGAATACTTGACCTAA
aceE          ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG
SEQ ID NO: 12 CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGTG
              TTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGCC
              CGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGCAA
              CTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCCGG
              GTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCTGG
              AACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCTCGA
              ACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCATTT
              ATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGCAG
              GATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCGGG
              CGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGAGC
              AGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCTCT
              TCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTCCC
              GACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCAGG
              CTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATACC
              TCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATGGA
              CGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTGAAA
              AACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAGCGT
              CTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGAACT
              GGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAAGTGA
              TGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATACCAGC
              GGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGCGACTA
              CCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGAACACT
              TCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAGACTGG
              ACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCACGATCC
              GAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAAACCAAAG
              GCAAAGCGACAGTAATCCTTGCTCATACCATTAAAGGTTACGGC
              ATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCACCAGGTTAA
              GAAAATGAACATGGACGGTGTGCGTCATATCCGCGACCGTTTCA
              ATGTGCCGGTGTCTGATGCAGATATCGAAAAACTGCCGTACATC
              ACCTTCCCGGAAGGTTCTGAAGAGCATACCTATCTGCACGCTCA
              GCGTCAGAAACTGCACGGTTATCTGCCAAGCCGTCAGCCGAACT
              TCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAGACTTCGGCGCG
              CTGTTGGAAGAGCAGAGCAAAGAGATCTCTACCACTATCGCTTT
              CGTTCGTGCTCTGAACGTGATGCTGAAGAACAAGTCGATCAAAG
              ATCGTCTGGTACCGATCATCGCCGACGAAGCGCGTACTTTCGGT
              ATGGAAGGTCTGTTCCGTCAGATTGGTATTTACAGCCCGAACGG
              TCAGCAGTACACCCCGCAGGACCGCGAGCAGGTTGCTTACTATA
              AAGAAGACGAGAAAGGTCAGATTCTGCAGGAAGGGATCAACGAG
              CTGGGCGCAGGTTGTTCCTGGCTGGCAGCGGCGACCTCTTACAG
              CACCAACAATCTGCCGATGATCCCGTTCTACATCTATTACTCGA
              TGTTCGGCTTCCAGCGTATTGGCGATCTGTGCTGGGCGGCTGGC
              GACCAGCAAGCGCGTGGCTTCCTGATCGGCGGTACTTCCGGTCG
              TACCACCCTGAACGGCGAAGGTCTGCAGCACGAAGATGGTCACA
              GCCACATTCAGTCGCTGACTATCCCGAACTGTATCTCTTACGAC
              CCGGCTTACGCTTACGAAGTTGCTGTCATCATGCATGACGGTCT
              GGAGCGTATGTACGGTGAAAAACAAGAGAACGTTTACTACTACA
              TCACTACGCTGAACGAAAACTACCACATGCCGGCAATGCCGGAA
              GGTGCTGAGGAAGGTATCCGTAAAGGTATCTACAAACTCGAAAC
              TATTGAAGGTAGCAAAGGTAAAGTTCAGCTGCTCGGCTCCGGTT
              CTATCCTGCGTCACGTCCGTGAAGCAGCTGAGATCCTGGCGAAA
              GATTACGGCGTAGGTTCTGACGTTTATAGCGTGACCTCCTTCAC
              CGAGCTGGCGCGTGATGGTCAGGATTGTGAACGCTGGAACATGC
              TGCACCCGCTGGAAACTCCGCGCGTTCCGTATATCGCTCAGGTG
              ATGAACGACGCTCCGGCAGTGGCATCTACCGACTATATGAAACT
              GTTCGCTGAGCAGGTCCGTACTTACGTACCGGCTGACGACTACC
              GCGTACTGGGTACTGATGGCTTCGGTCGTTCCGACAGCCGTGAG
              AACCTGCGTCACCACTTCGAAGTTGATGCTTCTTATGTCGTGGT
              TGCGGCGCTGGGCGAACTGGCTAAACGTGGCGAAATCGATAAGA
              AAGTGGTTGCTGACGCAATCGCCAAATTCAACATCGATGCAGAT
              AAAGTTAACCCGCGTCTGGCGTAA
aceF          ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAGT
SEQ ID NO: 13 TGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGAAG
              CCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCTATG
              GAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAAAGT
              CTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGATTT
              TCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAGGCA
              GAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCGGCTGC
              GGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCGACGAAG
              TTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAAAGTTGAA
              GCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAGGCTTCTAT
              GGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGAGATCAAAG
              TGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTGATTATGGTC
              TTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCCGGCCGCTAA
              ACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCAGCGGCTGGCG
              TGAAAGAAGTTAACGTTCCGGATATCGGCGGTGACGAAGTTGAA
              GTGACTGAAGTGATGGTGAAAGTGGGCGACAAAGTTGCCGCTGA
              ACAGTCACTGATCACCGTAGAAGGCGACAAAGCTTCTATGGAAG
              TTCCGGCGCCGTTTGCAGGCGTCGTGAAGGAACTGAAAGTCAAC
              GTTGGCGATAAAGTGAAAACTGGCTCGCTGATTATGATCTTCGA
              AGTTGAAGGCGCAGCGCCTGCGGCAGCTCCTGCGAAACAGGAAG
              CGGCAGCGCCGGCACCGGCAGCAAAAGCTGAAGCCCCGGCAGCA
              GCACCAGCTGCGAAAGCGGAAGGCAAATCTGAATTTGCTGAAAA
              CGACGCTTATGTTCACGCGACTCCGCTGATCCGCCGTCTGGCAC
              GCGAGTTTGGTGTTAACCTTGCGAAAGTGAAGGGCACTGGCCGT
              AAAGGTCGTATCCTGCGCGAAGACGTTCAGGCTTACGTGAAAGA
              AGCTATCAAACGTGCAGAAGCAGCTCCGGCAGCGACTGGCGGTG
              GTATCCCTGGCATGCTGCCGTGGCCGAAGGTGGACTTCAGCAAG
              TTTGGTGAAATCGAAGAAGTGGAACTGGGCCGCATCCAGAAAAT
              CTCTGGTGCGAACCTGAGCCGTAACTGGGTAATGATCCCGCATG
              TTACTCACTTCGACAAAACCGATATCACCGAGTTGGAAGCGTTC
              CGTAAACAGCAGAACGAAGAAGCGGCGAAACGTAAGCTGGATGT
              GAAGATCACCCCGGTTGTCTTCATCATGAAAGCCGTTGCTGCAG
              CTCTTGAGCAGATGCCTCGCTTCAATAGTTCGCTGTCGGAAGAC
              GGTCAGCGTCTGACCCTGAAGAAATACATCAACATCGGTGTGGC
              GGTGGATACCCCGAACGGTCTGGTTGTTCCGGTATTCAAAGACG
              TCAACAAGAAAGGCATCATCGAGCTGTCTCGCGAGCTGATGACT
              ATTTCTAAGAAAGCGCGTGACGGTAAGCTGACTGCGGGCGAAAT
              GCAGGGCGGTTGCTTCACCATCTCCAGCATCGGCGGCCTGGGTA
              CTACCCACTTCGCGCCGATTGTGAACGCGCCGGAAGTGGCTATC
              CTCGGCGTTTCCAAGTCCGCGATGGAGCCGGTGTGGAATGGTAA
              AGAGTTCGTGCCGCGTCTGATGCTGCCGATTTCTCTCTCCTTCG
              ACCACCGCGTGATCGACGGTGCTGATGGTGCCCGTTTCATTACC
              ATCATTAACAACACGCTGTCTGACATTCGCCGTCTGGTGATGTA
              A
lpd           ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAGG
SEQ ID NO: 14 CCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC
              TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT
              TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT
              AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGTA
              TCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTACC
              TGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCTGG
              TATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGGGTA
              AATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAACGGC
              AAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCGGGTTC
              TCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTA
              TCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAA
              CGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGAAATGGG
              CACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGGTTGAAA
              TGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATCGTTAAA
              GTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGATGCTGGA
              AACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATG
              TGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTAC
              GACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAA
              CCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACGACCGTGGTT
              TCATCCGCGTTGACAAACAGCTGCGTACCAACGTACCGCACATC
              TTTGCTATCGGCGATATCGTCGGTCAACCGATGCTGGCACACAA
              AGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCCGGTA
              AGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTAT
              ACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGC
              GAAAGAGAAAGGCATCAGCTATGAAACCGCCACCTTCCCGTGGG
              CTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCAGACGGTATG
              ACCAAGCTGATTTTCGACAAAGAATCTCACCGTGTGATCGGTGG
              TGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGGTGAAATCG
              GCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTG
              ACCATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGC
              GGCAGAAGTGTTCGAAGGTAGCATTACCGACCTGCCGAACCCGA
              AAGCGAAGAAGAAGTAA
tesB          ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA
SEQ ID NO: 15 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG
              GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG
              TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC
              GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA
              TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC
              GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA
              TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC
              AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG
              GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT
              GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC
              CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA
              CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA
              CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA
              ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC
              GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT
              CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG
              AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG
              TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA
              AGGGGTGATGCGTAATCACAATTAA
acuI          ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT
SEQ ID NO: 16 CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGACG
              TTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGCC
              CTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT
              GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC
              ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG
              GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCG
              CGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACT
              TACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGATG
              TTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGTAA
              TGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCTCCG
              TTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCGGCA
              GTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTTGGG
              GGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAGGTAC
              GCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCG
              GGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTATCG
              CGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTGC
              CCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTGGCA
              GGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGCAGC
              GTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGG
              AGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGACAGCA
              CCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTATCCC
              CGTAACGCCCTAA

TABLE 3
Propionate Cassette Sequences Sleeping Beauty Operon
Sbm           ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAAT
SEQ ID NO: 17 TGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAAC
              CGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCGAT
              CTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCCGC
              CCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAACC
              GTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAGAG
              TCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAAAG
              GTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTACGA
              CTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCGGGC
              GTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCGACC
              AGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAATGG
              CGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCAGAA
              GAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATTCAAA
              ACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATATTTA
              CCCACCAAAACCGTCAATGCGCATTATCGCCGACATCATCGCC
              TGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGTATCA
              GCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTGCAGCA
              GGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCAAAGCA
              GCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCCTCGCC
              TGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGAACGT
              CGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGCGGTC
              AGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCTGCGTA
              CCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACAGGATCC
              GTATAACAACGTTATCCGCACCACCATTGAAGCGCTGGCTGCG
              ACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCTTTGACG
              AAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCATTGCCCG
              CAACACCCAGATCATCATCCAGGAAGAATCAGAACTCTGCCGC
              ACCGTCGATCCACTGGCCGGATCCTATTACATTGAGTCGCTGA
              CCGATCAAATCGTCAAACAAGCCAGAGCTATTATCCAACAGAT
              CGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAGCAGGTCTG
              CCAAAACGAATGATCGAAGAGGCCTCAGCGCGCGAACAGTCGC
              TGATCGACCAGGGCAAGCGTGTCATCGTTGGTGTCAACAAGTA
              CAAACTGGATCACGAAGACGAAACCGATGTACTTGAGATCGAC
              AACGTGATGGTGCGTAACGAGCAAATTGCTTCGCTGGAACGCA
              TTCGCGCCACCCGTGATGATGCCGCCGTAACCGCCGCGTTGAA
              CGCCCTGACTCACGCCGCACAGCATAACGAAAACCTGCTGGCT
              GCCGCTGTTAATGCCGCTCGCGTTCGCGCCACCCTGGGTGAAA
              TTTCCGATGCGCTGGAAGTCGCTTTCGACCGTTATCTGGTGCC
              AAGCCAGTGTGTTACCGGCGTGATTGCGCAAAGCTATCATCAG
              TCTGAGAAATCGGCCTCCGAGTTCGATGCCATTGTTGCGCAAA
              CGGAGCAGTTCCTTGCCGACAATGGTCGTCGCCCGCGCATTCT
              GATCGCTAAGATGGGCCAGGATGGACACGATCGCGGCGCGAAA
              GTGATCGCCAGCGCCTATTCCGATCTCGGTTTCGACGTAGATT
              TAAGCCCGATGTTCTCTACACCTGAAGAGATCGCCCGCCTGGC
              CGTAGAAAACGACGTTCACGTAGTGGGCGCATCCTCACTGGCT
              GCCGGTCATAAAACGCTGATCCCGGAACTGGTCGAAGCGCTGA
              AAAAATGGGGACGCGAAGATATCTGCGTGGTCGCGGGTGGCGT
              CATTCCGCCGCAGGATTACGCCTTCCTGCAAGAGCGCGGCGTG
              GCGGCGATTTATGGTCCAGGTACACCTATGCTCGACAGTGTGC
              GCGACGTACTGAATCTGATAAGCCAGCATCATGATTAA
ygfD          ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC
SEQ ID NO: 18 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGGT
              GGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGCTG
              CTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACTGG
              GCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTTGA
              GGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCGCG
              GTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAGCA
              TTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCCGA
              AGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGGGC
              GGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCGAAG
              CAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCGTCGG
              GCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTTTTATC
              TCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGGCATTA
              AAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATCAACAA
              AGACGATGGCGATAACCATACCAATGTCGCCATTGCCCGGCAT
              ATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAATACGACG
              AATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGGAAAAACG
              TGGAATCGATGAGATCTGGCACGCCATCATCGACTTCAAAACC
              GCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCGGCAACAAC
              AATCGGTGGAATGGCTGCGTAAGCAGACCGAAGAAGAAGTACT
              GAATCACCTGTTCGCGAATGAAGATTTCGATCGCTATTACCGC
              CAGACGCTTTTAGCGGTCAAAAACAATACGCTCTCACCGCGCA
              CCGGCCTGCGGCAGCTCAGTGAATTTATCCAGACGCAATATTT
              TGATTAA
ygfG          ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG
SEQ ID NO: 19 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG
              TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC
              AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG
              GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC
              GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT
              CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT
              CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT
              CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC
              TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG
              GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA
              AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG
              CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC
              TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA
              AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA
              CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGTA
              TTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTACCA
              GGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAATTTC
              GTTGGTCATTAA
ygfH          ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCAG
SEQ ID NO: 20 AAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTAC
              CCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCGC
              AGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAATTC
              GCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGATGT
              ACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATCAA
              ACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGGTGA
              GTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGTCAA
              TTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAAGCA
              TCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGCGGGA
              TCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAAGTGAT
              CATTGAACTCAATCACTATCACGATCCGCGCGTTGCAGAACTG
              GCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAATAGCG
              TGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGCTATGT
              GCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAAACCAAC
              TTGCCCGACGCCGGTAATATGCTGGATAAGCAAAATCCCATGT
              GCCAGCAGATTGCCGATAACGTGGTCACGTTCTTATTGCAGGA
              AATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCCGCTGCAA
              AGTGGCGTGGGCAATATCAATAATGCGGTAATGGCGCGTCTGG
              GGGAAAACCCGGTAATTCCTCCGTTTATGATGTATTCGGAAGT
              GCTACAGGAATCGGTGGTGCATTTACTGGAAACCGGCAAAATC
              AGCGGGGCCAGCGCCTCCAGCCTGACAATCTCGGCCGATTCCC
              TGCGCAAGATTTACGACAATATGGATTACTTTGCCAGCCGCAT
              TGTGTTGCGTCCGCAGGAGATTTCCAATAACCCGGAAATCATC
              CGTCGTCTGGGCGTCATCGCTCTGAACGTCGGCCTGGAGTTTG
              ATATTTACGGGCATGCCAACTCAACACACGTAGCCGGGGTCGA
              TCTGATGAACGGCATCGGCGGCAGCGGTGATTTTGAACGCAAC
              GCGTATCTGTCGATCTTTATGGCCCCGTCGATTGCTAAAGAAG
              GCAAGATCTCAACCGTCGTGCCAATGTGCAGCCATGTTGATCA
              CAGCGAACACAGCGTCAAAGTGATCATCACCGAACAAGGGATC
              GCCGATCTGCGCGGTCTTTCCCCGCTTCAACGCGCCCGCACTA
              TCATTGATAATTGTGCACATCCTATGTATCGGGATTATCTGCA
              TCGCTATCTGGAAAATGCGCCTGGCGGACATATTCACCACGAT
              CTTAGCCACGTCTTCGACTTACACCGTAATTTAATTGCAACCG
              GCTCGATGCTGGGTTAA

TABLE 4
Sequences of Propionate Cassette from Propioni Bacteria
Description   Sequence
mutA          ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGACG
SEQ ID NO: 21 ACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCAA
              GGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTATTC
              AACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGAGT
              GTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGACAT
              CGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTGGGT
              TACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGCGCA
              ACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACGAGGA
              TCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAGACCTG
              GAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCCCGACG
              CGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGACGTCCT
              GCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACGACCAG
              GGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGCGCTCCG
              ACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCCTGGATCC
              CATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGGATCTGACC
              GTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTTCTCACCGG
              ACTCGCGCGCCGTCACGATCGACGCGAACGTCTACCACAACGC
              CGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCACTGGCCACC
              GGCGCGGAGTACGTGCGCGCCCTGGTCGAACAGGGCTTCAACG
              CCACAGAGGCCTTCGACACGATCAACTTCCGTGTCACCGCCAC
              CCACGACCAGTTCCTCACGATCGCCCGTCTTCGCGCCCTGCGC
              GAGGCATGGGCCCGCATCGGCGAGGTCTTTGGCGTGGACGAGG
              ACAAGCGCGGCGCTCGCCAGAATGCGATCACCAGTTGGCGTGA
              GCTCACCCGCGAAGACCCCTATGTCAACATCCTTCGCGGTTCG
              ATTGCCACCTTCTCCGCCTCCGTTGGCGGGGCCGAGTCGATCA
              CGACGCTGCCCTTCACCCAGGCCCTCGGCCTGCCGGAGGACGA
              CTTCCCGCTGCGCATCGCGCGCAACACGGGCATCGTGCTCGCC
              GAAGAGGTGAACATCGGCCGCGTCAACGACCCGGCCGGTGGCT
              CCTACTACGTCGAGTCGCTCACTCGCACCCTGGCCGACGCTGC
              CTGGAAGGAATTCCAGGAGGTCGAGAAGCTCGGTGGCATGTCG
              AAGGCGGTCATGACCGAGCACGTCACCAAGGTGCTCGACGCCT
              GCAATGCCGAGCGCGCCAAGCGCCTGGCCAACCGCAAGCAGCC
              GATCACCGCGGTCAGCGAGTTCCCGATGATCGGGGCCCGCAGC
              ATCGAGACCAAGCCGTTCCCAACCGCTCCGGCGCGCAAGGGCC
              TGGCCTGGCATCGCGATTCCGAGGTGTTCGAGCAGCTGATGGA
              TCGCTCCACCAGCGTCTCCGAGCGCCCCAAGGTGTTCCTTGCC
              TGCCTGGGCACCCGTCGCGACTTCGGTGGCCGCGAGGGCTTCT
              CCAGCCCGGTATGGCACATCGCCGGTATCGACACCCCGCAGGT
              CGAAGGCGGCACCACCGCCGAGATCGTCGAGGCGTTCAAGAAG
              TCGGGCGCCCAGGTGGCCGATCTCTGCTCGTCCGCCAAGATCT
              ACGCGCAGCAGGGACTTGAGGTTGCCAAGGCGCTCAAGGCCGC
              CGGCGCGAAGGCCCTGTATCTGTCGGGCGCCTTCAAGGAGTTC
              GGCGATGACGCCGCCGAGGCCGAGAAGCTGATCGACGGACGCC
              TGTACATGGGCATGGATGTCGTCGACACCCTGTCCTCCACCCT
              TGATATCTTGGGAGTCGCGAAGTGA
mutB          GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG
SEQ ID NO: 22 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC
              CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCAG
              ATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGACA
              TGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCCA
              CGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGATT
              CGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCCT
              TCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGGT
              TGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAAT
              CCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATCG
              ACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGCT
              GGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGCTG
              CCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGGCG
              TCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACATCCT
              CAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCGCAG
              CCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGAGTG
              CCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTACCA
              CATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGCCTAC
              ACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAGTCGG
              TGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCTTCTT
              CTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAAGCTG
              CGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAGTTCG
              GGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCACTCGCA
              GACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACAACAAC
              GTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCAGGGCC
              ATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGCCATTGC
              CCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAACACCCAG
              CTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTGATCGACC
              CGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACCTGGGACCT
              GGCCCGCAAGGCATGGGGCCACATCCAGGAGGTCGAGAAGGTC
              GGCGGCATGGCCAAGGCCATCGAAAAGGGCATCCCCAAGATGC
              GCATTGAGGAAGCCGCCGCCCGCACCCAGGCACGCATCGACTC
              CGGCCGTCAGCCGCTGATCGGCGTGAACAAGTACCGCCTGGAG
              CACGAGCCGCCGCTCGATGTGCTCAAGGTTGACAACTCCACGG
              TGCTCGCCGAGCAGAAGGCCAAGCTGGTCAAGCTGCGCGCCGA
              GCGCGATCCCGAGAAGGTCAAGGCCGCCCTCGACAAGATCACC
              TGGGCTGCCGCCAACCCCGACGACAAGGATCCGGATCGCAACC
              TGCTGAAGCTGTGCATCGACGCTGGCCGCGCCATGGCGACGGT
              CGGCGAGATGAGCGACGCGCTCGAGAAGGTCTTCGGACGCTAC
              ACCGCCCAGATTCGCACCATCTCCGGTGTGTACTCGAAGGAAG
              TGAAGAACACGCCTGAGGTTGAGGAAGCACGCGAGCTCGTTGA
              GGAATTCGAGCAGGCCGAGGGCCGTCGTCCTCGCATCCTGCTG
              GCCAAGATGGGCCAGGACGGTCACGACCGTGGCCAGAAGGTCA
              TCGCCACCGCCTATGCCGACCTCGGTTTCGACGTCGACGTGGG
              CCCGCTGTTCCAGACCCCGGAGGAGACCGCACGTCAGGCCGTC
              GAGGCCGATGTGCACGTGGTGGGCGTTTCGTCGCTCGCCGGCG
              GGCATCTGACGCTGGTTCCGGCCCTGCGCAAGGAGCTGGACAA
              GCTCGGACGTCCCGACATCCTCATCACCGTGGGCGGCGTGATC
              CCTGAGCAGGACTTCGACGAGCTGCGTAAGGACGGCGCCGTGG
              AGATCTACACCCCCGGCACCGTCATTCCGGAGTCGGCGATCTC
              GCTGGTCAAGAAACTGCGGGCTTCGCTCGATGCCTAG
GI:18042134   ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT
SEQ ID NO: 23 ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGGA
              GACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGGAG
              CAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGCTGA
              CCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAACGA
              CGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGGTCGC
              GCCGGACTGCACCACATGGCATGGCGTGTCGATGACATCGACG
              CCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTGCTGTA
              TGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATCAACTTC
              ATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGCTCACCC
              AGTACCCGAAGAACTGA
mmdA          ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAAG
SEQ ID NO: 24 GTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAAGC
              CGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAGGGT
              AAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGATCCCC
              ATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCACCAC
              GTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGGCGTG
              GTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCACGCCG
              CGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCGAGAC
              GCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGCTGCTC
              ACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGGCGCCC
              GGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGGCAAGAT
              GTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCGCAGATC
              GCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGTATTCGC
              CGGCACTGACTGACTTCATCATCATGACCAAGAAGGCCCATAT
              GTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACCGGCGAG
              GATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCCCATATGG
              CCATCTCGGGCAATATCCACTTCGTGGCCGAGGACGACGACGC
              CGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCTTCCGCAG
              AACAACACTGAGGAAGCATCCTTCGTCAACCCGAACAATGACG
              TCAGCCCCAATACCGAGCTGCGCGACATCGTTCCGATTGACGG
              CAAGAAGGGCTATGACGTGCGCGATGTCATTGCCAAGATCGTC
              GACTGGGGTGACTACCTCGAGGTCAAGGCCGGCTATGCCACCA
              ACCTCGTGACCGCCTTCGCCCGGGTCAATGGTCGTTCGGTGGG
              CATCGTGGCCAATCAGCCGTCGGTGATGTCGGGTTGCCTCGAC
              ATCAACGCCTCTGACAAGGCCGCCGAATTCGTGAATTTCTGCG
              ATTCGTTCAACATCCCGCTGGTGCAGCTGGTCGACGTGCCGGG
              CTTCCTGCCCGGCGTGCAGCAGGAGTACGGCGGCATCATTCGC
              CATGGCGCGAAGATGCTGTACGCCTACTCCGAGGCCACCGTGC
              CGAAGATCACCGTGGTGCTCCGCAAGGCCTACGGCGGCTCCTA
              CCTGGCCATGTGCAACCGTGACCTTGGTGCCGACGCCGTGTAC
              GCCTGGCCCAGCGCCGAGATTGCGGTGATGGGCGCCGAGGGTG
              CGGCAAATGTGATCTTCCGCAAGGAGATCAAGGCTGCCGACGA
              TCCCGACGCCATGCGCGCCGAGAAGATCGAGGAGTACCAGAAC
              GCGTTCAACACGCCGTACGTGGCCGCCGCCCGCGGTCAGGTCG
              ACGACGTGATTGACCCGGCTGATACCCGTCGAAAGATTGCTTC
              CGCCCTGGAGATGTACGCCACCAAGCGTCAGACCCGCCCGGCG
              AAGAAGCATGGAAACTTCCCCTGCTGA
PFREUD_18870  ATGAGTCCGCGAGAAATTGAGGTTTCCGAGCCGCGCGAGGTTG
SEQ ID NO: 25 GTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGAT
              GGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTGCA
              GACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGGTG
              GTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGATCC
              TTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACAGC
              CGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTACC
              GCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAGTC
              CGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCATG
              AATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAAGA
              AGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGATCAG
              CCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGTCAG
              CTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACATGG
              CCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAAGGC
              CATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCTGCAC
              TGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATGAAGG
              CCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATCTCGTC
              CATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGGTTGCC
              GAGATGCTCGAGGGCACCGGGTACACCACCAACCTTGACTACG
              ATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCATCCGCCC
              GAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGACACCTCG
              ATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCCAACATGG
              AGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAGATGGACGA
              GGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCCGCCGGCTTC
              CCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCGGCACGCAGG
              CCGTGTTCAACGTGATGATGGGCGAGTACAAGAGGATGACCGG
              CGAGTTCGCCGACATCATGCTCGGCTACTACGGCGCCAGCCCG
              GCCGATCGCGATCCGAAGGTGGTCAAGTTGGCCGAGGAGCAGT
              CCGGCAAGAAGCCGATCACCCAGCGCCCGGCCGATCTGCTGCC
              CCCCGAGTGGGAGGAGCAGTCCAAGGAGGCCGCGGCCCTCAAG
              GGCTTCAACGGCACCGACGAGGACGTGCTCACCTATGCACTGT
              TCCCGCAGGTCGCTCCGGTCTTCTTCGAGCATCGCGCCGAGGG
              CCCGCACAGCGTGGCTCTCACCGATGCCCAGCTGAAGGCCGAG
              GCCGAGGGCGACGAGAAGTCGCTCGCCGTGGCCGGTCCCGTCA
              CCTACAACGTGAACGTGGGCGGAACCGTCCGCGAAGTCACCGT
              TCAGCAGGCGTGA
Bccp          ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTTG
SEQ ID NO: 26 ACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACCAT
              CCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGCGCA
              GCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGAGATTC
              CCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTGAAGGA
              GGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTTCTCGAG
              GCCATGAAGATGGAGACCGAGATCAACGCTCCCACCGACGGCA
              AGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCCGTGCAGGG
              CGGTCAGGGTCTCATCAAGATCGGCTGA

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof.

Table 5 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria.

TABLE 5
Polypeptide Sequences for Propionate Synthesis
Pct         MRKVPIITADEAAKLIKDGDTVTTSGFVGNAIPEALDRAVEKRFLET
SEQ ID      GEPKNITYVYCGSQGNRDGRGAEHFAHEGLLKRYIAGHWATVPALGK
NO: 27      MAMENKMEAYNVSQGALCHLFRDTASHKPGVFTKVGIGTFIDPRNGG
            GKVNDITKEDIVELVEIKGQEYLFYPAFPIHVALIRGTYADESGNIT
            FEKEVAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRHVKVPGI
            YVDYVVVADPEDHQQSLDCEYDPALSGEHRRPEVVGEPLPLSAKKVI
            GRRGAIELEKDVAVNLGVGAPEYVASVADEEGIVDFMTLTAESGAIG
            GVPAGGVRFGASYMADALIDQGYQFDYYDGGGLDLCYLGLAECDEKG
            NINVSRFGPRIAGCGGFINITQNTPKVFFCGTFTAGGLKVKIEDGKV
            IIVQEGKQKKFLKAVEQITFMGDVALANKQQVTYITERCVFLLKEDG
            LHLSEIAPGIDLQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLM
            GLKEMKS*
lcdA        MSLTQGMKAKQLLAYFQGKADQDAREAKARGELVCWSASVAPPEFCV
SEQ ID      TMGIAMIYPETHAAGIGARKGAMDMLEVADRKGYNVDCCSYGRVNMG
NO: 28      YMECLKEAAITGVKPEVLVNSPAADVPLPDLVITCNNICNTLLKWYE
            NLAAELDIPCIVIDVPFNHTMPIPEYAKAYIADQFRNAISQLEVICG
            RPFDWKKFKEVKDQTQRSVYHWNRIAEMAKYKPSPLNGFDLFNYMAL
            IVACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAV
            WPHLGHTFKSMKNLNSIMTGTAYPALWDLHYDANDESMHSMAEAYTR
            IYINTCLQNKVEVLLGIMEKGQVDGTVYHLNRSCKLMSFLNVETAEI
            IKEKNGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE*
lcdB        MSRVEAILSQLKDVAANPKKAMDDYKAETGKGAVGIMPIYSPEEMVH
SEQ ID      AAGYLPMGIWGAQGKTISKARTYLPAFACSVMQQVMELQCEGAYDDL
NO: 29      SAVIFSVPCDTLKCLSQKWKGTSPVIVFTHPQNRGLEAANQFLVTEY
            ELVKAQLESVLGVKISNAALENSIAIYNENRAVMREFVKVAADYPQV
            IDAVSRHAVFKARQFMLKEKHTALVKELIAEIKATPVQPWDGKKVVV
            TGILLEPNELLDIFNEFKIAIVDDDLAQESRQIRVDVLDGEGGPLYR
            MAKAWQQMYGCSLATDTKKGRGRMLINKTIQTGADAIVVAMMKFCDP
            EEWDYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML*
lcdC        MYTLGIDVGSASSKAVILKDGKDIVAAEVVQVGTGSSGPQRALDKAFE
SEQ ID      VSGLKKEDISYTVATGYGRFNFSDADKQISEISCHAKGIYFLVPTART
NO: 30      IIDIGGQDAKAIRLDDKGGIKQFFMNDKCAAGTGRFLEVMARVLETTL
            DEMAELDEQATDTAPISSTCTVFAESEVISQLSNGVSRNNIIKGVHLS
            VASRACGLAYRGGLEKDVVMTGGVAKKAGVVRAVAGVLKTDVIVAPNP
            QTTGALGAALYAYEAAQKKX
etfA        MAFNSAD1NSFRDIWVFCEQREGKLINTDFELISEGRKLADERGSKL
SEQ ID      VGILLGHEVEEIAKELGGYGADKVIVCDHPELKFYTTDAYAKVLCDV
NO: 31      VMEEKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDHNKYV
            DFLSTSSTLDISSMTFPMEDTNLKMTRPAFGGHLMATIICPRFRPCM
            STVRPGVMKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETK
            KIVDLIGAEIIVSVGRGISKDVQGGIALAEKLADAFGNGVVGGSRAV
            IDSGWLPADHQVGQTGKTVHPKVYVALGISGAIQHKAGMQDSELIIA
            VNKDETAPIFDCADYGITGDLFKIVPMMIDAIKEGKMA*
acrB        MRIYVCVKQVPDTSGKVAVHPDGTLNRASMAAIINPDDMSAIEQALK
SEQ ID      LKDETGCQVTALTMGPPPAEGMLREIIAMGADDGVLISAREFGGSDT
NO: 32      FATSQIISAAIHKLGLSNEDMIFCGRQAIDGDTAQVGPQIAEKLSIP
            QVTYGAGIKKSGDLVLVKRMLEDGYMMIEVETPCLITCIQDKAVKPR
            YMTLNGIMECYSKPLLVLDYEALKDEPLIELDTIGLKGSPTNIFKSF
            TPPQKGVGVMLQGTDKEKVEDLVDKLMQKHVI*
acrC        MFLLKTKKERMKRMDFSLTREQEMLKKLARQFAEIELEPVAEEIDRE
SEQ ID      HVFPAENFKKMAEIGLTGIGIPKEFGGSGGGTLEKVIAVSEFGKKCM
NO: 33      ASASILSIHLIAPQAIYKYGTKEQKETYLPRLTKGGELGAFALTEPN
            AGSDAGAVKTTAILDSQTNEYVLNGTKCFISGGGRAGVLVIFALTEP
            KKGLKGMSAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPA
            ANLLGKEGKGFKIAMEALDGARIGVGAQAIGIAEGAIDLSVKYVHER
            IQFGKPIANLQGIQWYIADMATKTAAARALVEFAAYLEDAGKPFTKE
            SAMCKLNASENARFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIY
            EGTSEIHKWAREVMKR*
thrAfbr     MRVLKFGGTSVANAERFLRVADILESKARQGQVATVLSAPAKITNHL
SEQ ID      VAMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKIFV
NO: 34      DQEFAQIKHVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEAR
            GHMVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIFADHMVLMA
            GFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTC
            DPRQVPDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLI
            KNTGNPQAPGTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMV
            GMAARVFAAMSRARISVVLITQSSSEYSISFCVPQSDCVRAERAMQE
            EFYLELKEGLLEPLAVTERLAIISSSGDGMRTLRGISAKFFAALARA
            NINIVAIAQRSSERSISSSVNNDDATTGVRVTHQMLFNTDQVIEVFV
            IGVGGVGGALLEQLKRQQSWLKNKHIDLRVCGVANSKALLTNVHGLN
            LENWQEELAQAKEPFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQY
            ADFLREGFHSSTPNKKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGA
            GLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGMSFSEATT
            LAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVL
            PAEFNAEGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDED
            GVCRVKIAEVDGNDPLFKVKNGENALAFYSHYYQPLPLVLRGYGAGN
            DVTAAGVFADLLRTLSWKLGV*
thrB        MVKVYAPASSANMSVGFDVLGAAVTPVDGALLGDVVTVEAAETFSLN
SEQ ID      NLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS
NO: 35      GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD
            NVAPCFLGGMQLMIEENDIISQQVPGFDEWLWVLAYPGIKVSTAEAR
            AILPAQYRRQDCIAHGRHLAGFIHACYSRQPELAAKLMKDVIAEPYR
            ERLLPGFRQARQAVAEIGAVASGISGSGPTLFALCDKPETAQRVADW
            LGKNYLQNQEGFVHICRLDTAGARVLEM*
thrC        MKLYNLKDHNEQVSFAQAVTQGLGKNQGLFFPHDLPEFSLTEIDEML
SEQ ID      KLDFVTRSAKILSAFIGDEIPQEILEERVRAAFAFPAPVANVESDVG
NO: 36      CLELFHGPTLAFKDFGGRFMAQMLTHIAGDKPVTILTATSGDTGAAV
            AHAFYGLPNVKVVILYPRGKISPLQEKLFCTLGGNIETVAIDGDFDA
            CQALVKQAFDDEELKVALGLNSANSINISRLLAQICYYFEAVAQLPQ
            ETRNQLVVSVPSGNFGDLTAGLLAKSLGLPVKRFIAATNVNDTVPRF
            LHDGQWSPKATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYA
            AVDDETTQQTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGT
            AHPAKFKESVEAILGETLDLPKELAERADLPLLSHNLPADFAALRKL
            MMNHQ*
ilvAfbr     MSETYVSEKSPGVMASGAELIRAADIQTAQARISSVIAPTPLQYCPR
SEQ ID      LSEETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAAS
NO: 37      AGNHAQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVV
            TGNNFDEASAAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILSQL
            TSMGKSADHVMVPVGGGGLLAGVVSYMADMAPRTAIVGIEPAGAASM
            QAALRNGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVHMMSA
            TEGAVCTEMLDLYQNEGIIAEPAGALSIAGLKEMSFAPGSAVVCIIS
            GGNNDVLRYAEIAERSLVHRGLKHYFLVNFPQKPGQLRHFLEDILGP
            DDDITLFEYLKRNNRETGTALVGIHLSEASGLDSLLERMEESAIDSR
            RLEPGTPEYEYLT*
ace         MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKG
SEQ ID      GVNVAAGTG1SNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTV
NO: 38      LRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQ
            GHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFW
            QFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMD
            EPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGI
            FEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSK
            DGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKK
            AQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRD
            RFNVPVSDADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNF
            TEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLV
            PIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQ
            ILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDL
            CWAAGDQQARGFLIGGTSGRTTLNGEGLQHEDGHSHIQSLTIPNCIS
            YDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEG
            AEEGIRKGIYKLETIEGSKGKVQLLGSGSXLRHVREAAEILAKDYGV
            GSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAV
            ASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSREMLRHHFEVDA
            SYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA*
aceF        MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVP
SEQ ID      SPQAGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEA
NO: 39      APAAAPAAAAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVE
            GDKASMEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAP
            AAKQEAAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGDKVAAE
            QSLITVEGDKASMEVPAPFAGVVKELKVNVGDKVKTGSLIMIFEVEG
            AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHA
            TPLIRRLAREFGVNLAKVKGTGRKGRILREDVQAYVKEAIKRAEAAP
            AATGGGIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMI
            PHVTHFDKTDITELEAFRKQQNEEAAKRKLDVKITPVVFIMKAVAAA
            LEQMPRFNSSLSEDGQRLTLKKYINIGVAVDIPNGLVVPVFKDVNKK
            GIIELSRELMTISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPI
            VNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADG
            ARFITIINNTLSDIRRLVM*
Lpd         MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLN
SEQ ID      VGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVI
NO: 40      NQLTGGLAGMAKGRKVKVVMGLGKFTGANTLEVEGENGKTVINFDNA
            IIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGL
            EMGTVYHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLE
            TKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDA
            GKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGH
            VAAEVIAGKKHYFDPKVIPSIAYTKPEVAWVGLTEKEAKEKGISYET
            ATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLG
            EIGLAIEMGCDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPK
            AKKK*
tesB        MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA
SEQ ID      KETVPEERLVHSFHSYFLRPGDSKKPIIYDVETLRDGNSFSARRVAA
NO: 41      IQNGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLA
            HLLPPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQVWIRANGSV
            PDDLRVHQYLLGYASDLNFLPVALQPHGIGFLEPGIQIATIDHSMWF
            HRPFNLNEWLLYSVESTSASSARGFVRGEFYTQDGVLVASTVQEGVM
            RNKN*
acuI        MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYKDALAI
SEQ ID      TGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVILNGWGVGEKH
NO: 42      WGGLAERARVRGDWLVPLPAPLDLRQAAMIGTAGYTAMLCVLALERH
            GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR
            GLGAASVIDRNELTGKVRPLGQERWAGGIDVAGSTVLANMLSMMKYR
            GVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAWAR
            LASDLDPAKLEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP*
Sbm         MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL
SEQ ID      EVTGTLPGLPPYVRGPRATMYTAQPWTIRQYAGFSTAKESNAFYRRN
NO: 43      LAAGQKGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL
            FDQIPLDKMSVSMTMNGAVLPVLAFYIVAAEEQGVTPDKLTGTIQND
            ILKEYLCRNTYIYPPKPSMRIIADIIAWCSGNMPRFNTISISGYHMG
            EAGANCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMD
            LFMNVAMLRAARYLWSEAVSGFGAQDFKSLALRTHCQTSGWSLTEQD
            PYNNVIRTTIEALAATLGGTQSLHTNAFDEALGLPTDFSARIARNTQ
            IIIQEESELCRTVDPLAGSYYIESLTDQIVKQARAIIQQIDEAGGMA
            KAIEAGLPKRMIEEASAREQSLIDQGKRVIVGVNKYKLDHEDETDVL
            EIDNVMVRNEQIASLERIRATRDDAAVTAALMALTHAAQHNENLLAA
            AVNAARVRATLGEISDALEVAFDRYLVPSQCVTGVIAQSYHQSEKSA
            SEFDATVAQTEQFLADNGRRPRILIAKMGQDGHDRGAKVIASAYSDL
            GFDVDLSPMFSTPEEIARLAVENDVHVVGASSLAAGHKTLIPELVEA
            LKKWGREDICVVAGGVIPPQDYAFLQERGVAAIYGPGTPMLDSVRDV
            LNLISQHHD*
ygfD        MINEATLAESIRRLRQGERATLAQAMTLVESRHPRHQALSTQLLDAI
SEQ ID      MPYCGNTLRLGVTGTPGAGKSTFLEAFGMLLIREGLKVAVIAVDPSS
NO: 44      PVTGGSILGDKTRMNDLARAEAAFIRPVPSSGHLGGASQRARELMLL
            CEAAGYDVVIVETVGVGQSETEVARMVDCFISLQIAGGGDDLQGIKK
            GLMEVADLIVINKDDGDNHTNVAIARHMYESALHILRRKYDEWQPRV
            LTCSALEKRGIDEIWHAIIDFKTALTASGRLQQVRQQQSVEWLRKQT
            EEEVLNHLFANEDFDRYYRQTLLAVKNNTLSPRTGLRQLSEFIQTQY
            FD*
ygfG        MSYQYVNVVTINKVAVIEFNYGRKLNALSKVFIDDLMQALSDLNRPE
SEQ ID      IRCIILRAPSGSKVFSAGHDIHELPSGGRDPLSYDDPLRQITRMIQK
NO: 45      FPKPIISMVEGSVWGGAFEMIMSSDLIIAASTSTFSMTPVNLGVPYN
            LVGIHNLTRDAGFHIVKELIFTASPITAQRALAVGILNHVVEVEELE
            DFTLQMAHHISEKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGMRR
            AVYDSEDYQEGMNAFLEKRKPNFVGH*
yghH        METQWTRMTANEAAEIIQHNDMVAFSGFTPAGSPKALPTAIARRANE
SEQ ID      QHEAKKPYQIRLLTGASISAAADDVLSDADAVSNRAPYQTSSGLRKK
NO: 46      INQGAVSFVDLHLSEVAQMVNYGFFGDIDVAVIEASALAPDGRVWLT
            SGIGMAPTVVLLRAKKVHELNHYHDPRVAELADIVIPGAPPRRNSVS
            IFHAMDRVGTRYVQIDPKKIVAVVETNLPDAGNMLDKQNPMCQQIAD
            NVVTFLLQEMAHGRIPPEFLPLQSGVGNINNAVMARLGENPVIPPFM
            MYSEVLQESVVHLLETGKISGASASSLTISADSLRKIYDNMDYFASR
            IVLRPQEISNNPEIIRRLGVIALNVGLEFDIYGHANSTHVAGVDLMN
            GIGGSGDFERNAYLSIFMAPSIAKEGKISTVVPMCSRVDHSEHSVKV
            IITEQGIADLRGLSPLQRARTIIDNCAHPMYRDYLHRYLENAPGGHI
            HHDLSHVFDLHRNLIATGSMLG*
mutA        MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVEKVFNRGRP
SEQ ID      PEKQLTFAECLKRLTVHTVDGIDIVPMYRPKDAPKKLGYPGVTPFTRG
NO: 47      TTVRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDA
            IAPEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKD
            LALNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDANV
            YHNAGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATH
            DQFLTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDP
            YVNILRGSIATFSASVGGAESITTLPFTQALGLPEDDFPLRIARNTGI
            VLAEEVNIGRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMSKA
            VMTEHVTKVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFP
            TAPARKGLAWHRDSEVFEQLMDRSTSVSERPKVFLACLGTRRDFGGRE
            GFSSPVWHIAGIDTPQVEGGTTAEIVEAFKKSGAQVADLCSSAKIYAQ
            QGLEVAKALKAAGAKALYLSGAFKEFGDDAAEAEKLIDGRLYMGMDVV
            DTLSSTLDILGVAK
mutB
SEQ ID      VSTLPRFDSVDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGT
NO: 48      LFNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFSTAK
            ESNAFYRRNLAAGQKGLSVAFDLPTHRGYDSDNPRVAGDVGMAGVAID
            SIYDMRELFAGIPLDQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQL
            AGTIQNDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISI
            SGYHMQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQFAPRLSFFW
            GIGMNFFMEVAKLRAARMLWAKLVHQFGPKNPKSMSLRTHSQTSGWSL
            TAQDVYNNVVRTCIEAMAATQGHTQSLHTNSLDEAIALPTDFSARIAR
            NTQLFLQQESGTTRVIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGG
            MAKAIEKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPLDV
            LKVDNSTVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPD
            RNLLKLCIDAGRAMATVGEMSDALEKVFGRYTAQIRTISGVYSKEVKN
            TPEVEEARELVEEFEQAEGRRPRILLAKMGQDGHDRGQKVIATAYADL
            GFDVDVGPLFQTPEETARQAVEADVHVVGVSSLAGGHLTLVPALRKEL
            DKLGRPDILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVK
            KLRASLDA
GI:18042134 MSNEDLFICIDHVAYACPDADEASKYYQETFGWHELHREENPEQGVVE
SEQ ID      IMMAPAAKLTEHMTQVQVMAPLNDESTVAKWLAKHNGRAGLHHMAWRV
NO: 49      DDIDAVSATLRERGVQLLYDEPKLGTGGNRINFMHPKSGKGVLIELTQ
            YPKN
mmdA        MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHSQGKQTAR
SEQ ID      ERLNNLLDPHSFDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILGR
NO: 50      PVHAASQDFTVMGGSAGETQSTKVVETMEQALLTGTPFLFFYDSGGAR
            IQEGIDSLSGYGKMFFANVKLSGVVPQIAIIAGPCAGGASYSPALTDF
            IIMTKKAHMFITGPQVIKSVTGEDVTADELGGAEAHMAISGNIHFVAE
            DDDAAELIAKKLLSFLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGK
            KGYDVRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRSVGIVANQP
            SVMSGCLDINASDKAAEFVNFCDSFNIPLVQLVDVPGFLPGVQQEYGG
            IIRHGAKMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAW
            PSAEIAVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYV
            AAARGQVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC
PFREUD_     MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA
18870       GYWSVECWGGATYDSCIRFLNEDPWERLRTFRKLMPNSRLQMLLRGQN
SEQ ID      LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAVKK
NO: 51      AGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALLKP
            QPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVDT
            AISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPK
            YKKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVP
            RVRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYG
            ASPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGF
            NGTDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKS
            LAVAGPVTYNVNVGGTVREVTVQQA
Bccp        MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGAG
SEQ ID      AGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINA
NO: 52      PTDGKVEKVLVKERDAVQGGQGLIKIG

In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 5 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment or variant thereof. In some embodiments, genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of one or more polypeptide sequence of Table 5 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment thereof.

In one embodiment, the bacterial cell comprises a heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.

Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Clostridium propionicum, Megasphaera elsdenii, or Prevotella ruminicola.

In one embodiment, the propionate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the propionate gene cassette has been codon-optimized for use in Lactococcus. When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat liver disease, such as nonalcoholic steatohepatitis (NASH).

The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” relates to an element having qualitative biological activity in common with the wild-type enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N-terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.

As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including “homology,” is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).

In some embodiments, a propionate biosynthesis enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the propionate biosynthesis gene cassette is from Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionicum. In another embodiment, the propionate biosynthesis gene cassette is from a Megasphaera spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In another embodiment, the propionate biosynthesis gene cassette is from Prevotella spp. In one embodiment, the Prevotella spp. is Prevotella ruminicola. Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.

In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA^fbr, thrB, thrC, ilvA^fbr, aceE, aceF, and lpd, and optionally further comprise tesB. The genes may be codon-optimized, and translational and transcriptional elements may be added.

In one embodiment, the pct gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the pct gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In another embodiment, the pct gene comprises the sequence of SEQ ID NO: 1. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 1.

In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 95% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO: 2.

In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the lcdB gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the lcdB gene has at least about 95% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the lcdB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO: 3.

In one embodiment, the lcdC gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the lcdC gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the lcdC gene has at least about 95% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO: 4.

In one embodiment, the e0 gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the e0 gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 95% identity with SEQ ID NO: 5. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Accordingly, in one embodiment, the etfA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another embodiment, the e0 gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 5.

In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 6.

In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 7.

In one embodiment, the thrA^fbr gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the thrA^fbr gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the thrA^fbr gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the thrA^fbr gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the thrA^fbr gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. Accordingly, in one embodiment, the thrg^br gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In another embodiment, the thrA^fbr gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the thrA^fbr gene consists of the sequence of SEQ ID NO: 8.

In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 9.

In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 10.

In one embodiment, the ilvA^fbr gene has at least about 80% identity with SEQ ID NO: 11. In another embodiment, the ilvA^fbr gene has at least about 85% identity with SEQ ID NO: 11. In one embodiment, the ilvA^fbr gene has at least about 90% identity with SEQ ID NO: 11. In one embodiment, the ilvA^fbr gene has at least about 95% identity with SEQ ID NO: 11. In another embodiment, the ilvA^fbr gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. Accordingly, in one embodiment, the ilvA^fbr gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In another embodiment, the ilvA^fbr gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the ilvA^fbr gene consists of the sequence of SEQ ID NO: 11.

In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. Accordingly, in one embodiment, the aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 12. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 12.

In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. Accordingly, in one embodiment, the aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 13. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 13.

In one embodiment, the lpd gene has at least about 80% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 85% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 90% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 95% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. Accordingly, in one embodiment, the lpd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. In another embodiment, the lpd gene comprises the sequence of SEQ ID NO: 14. In yet another embodiment the lpd gene consists of the sequence of SEQ ID NO: 14.

In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15.

In one embodiment, the acuI gene has at least about 80% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 85% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 90% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 95% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. Accordingly, in one embodiment, the acuI gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. In another embodiment, the acuI gene comprises the sequence of SEQ ID NO: 16. In yet another embodiment the acuI gene consists of the sequence of SEQ ID NO: 16.

In one embodiment, the sbm gene has at least about 80% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.0. Accordingly, in one embodiment, the sbm gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 17. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 17.

In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.. Accordingly, in one embodiment, the ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 18. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 18.

In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.. Accordingly, in one embodiment, the ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 19. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 19.

In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20.. Accordingly, in one embodiment, the ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 20. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 20.

In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ ID NO: 27 through SEQ ID NO: 52.

In some embodiments, one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. glutamicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a R. sphaeroides propionate biosynthesis gene. The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.

In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate. In some embodiments, one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production. In some embodiments, the local production of propionate reduces food intake and ameliorates metabolic disease (Lin et al., 2012). In some embodiments, the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the propionate gene cassette is directly operably linked to a first promoter. In another embodiment, the propionate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the propionate gene cassette is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the propionate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the propionate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the propionate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

The propionate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the propionate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the propionate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located on a plasmid in the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the propionate gene cassette is expressed on a low-copy plasmid. In some embodiments, the propionate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of propionate.

Butyrate

In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 3). Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd2, eff133, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etfl33, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

Table 6 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.

TABLE 6
Exemplary Butyrate Cassette Sequences
Description   Sequence
bcd2          ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATA
SEQ ID NO: 53 TGTAAGCTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAAC
              TTGATGAAGAAGAAAGATTTCCTTATGAAACAGTGGAAAAAATG
              GCAAAAGCAGGAATGATGGGTATACCATATCCAAAAGAATATGG
              TGGAGAAGGTGGAGACACTGTAGGATATATAATGGCAGTTGAAG
              AATTGTCTAGAGTTTGTGGTACTACAGGAGTTATATTATCAGCT
              CATACATCTCTTGGCTCATGGCCTATATATCAATATGGTAATGA
              AGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGAAA
              AATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGAT
              GCGTCTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATA
              CATACTTAATGGCTCAAAAATATTTATAACAAACGCAATAGCTG
              GTGACATATATGTAGTAATGGCAATGACTGATAAATCTAAGGGG
              AACAAAGGAATATCAGCATTTATAGTTGAAAAAGGAACTCCTGG
              GTTTAGCTTTGGAGTTAAAGAAAAGAAAATGGGTATAAGAGGTT
              CAGCTACGAGTGAATTAATATTTGAGGATTGCAGAATACCTAAA
              GAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAGCAAT
              GTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTT
              TAGGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTA
              AAAGAAAGAGTACAATTTGGTAGACCATTATCAAAATTCCAAAA
              TACACAATTCCAATTAGCTGATATGGAAGTTAAGGTACAAGCGG
              CTAGACACCTTGTATATCAAGCAGCTATAAATAAAGACTTAGGA
              AAACCTTATGGAGTAGAAGCAGCAATGGCAAAATTATTTGCAGC
              TGAAACAGCTATGGAAGTTACTACAAAAGCTGTACAACTTCATG
              GAGGATATGGATACACTCGTGACTATCCAGTAGAAAGAATGATG
              AGAGATGCTAAGATAACTGAAATATATGAAGGAACTAGTGAAGT
              TCAAAGAATGGTTATTTCAGGAAAACTATTAAAATAG
etfB3         ATGAATATAGTCGTTTGTATAAAACAAGTTCCAGATACAACAGA
SEQ ID NO: 54 AGTTAAACTAGATCCTAATACAGGTACTTTAATTAGAGATGGAG
              TACCAAGTATAATAAACCCTGATGATAAAGCAGGTTTAGAAGAA
              GCTATAAAATTAAAAGAAGAAATGGGTGCTCATGTAACTGTTAT
              AACAATGGGACCTCCTCAAGCAGATATGGCTTTAAAAGAAGCTT
              TAGCAATGGGTGCAGATAGAGGTATATTATTAACAGATAGAGCA
              TTTGCGGGTGCTGATACTTGGGCAACTTCATCAGCATTAGCAGG
              AGCATTAAAAAATATAGATTTTGATATTATAATAGCTGGAAGAC
              AGGCGATAGATGGAGATACTGCACAAGTTGGACCTCAAATAGCT
              GAACATTTAAATCTTCCATCAATAACATATGCTGAAGAAATAAA
              AACTGAAGGTGAATATGTATTAGTAAAAAGACAATTTGAAGATT
              GTTGCCATGACTTAAAAGTTAAAATGCCATGCCTTATAACAACT
              CTTAAAGATATGAACACACCAAGATACATGAAAGTTGGAAGAAT
              ATATGATGCTTTCGAAAATGATGTAGTAGAAACATGGACTGTAA
              AAGATATAGAAGTTGACCCTTCTAATTTAGGTCTTAAAGGTTCT
              CCAACTAGTGTATTTAAATCATTTACAAAATCAGTTAAACCAGC
              TGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAATTA
              TCATAGATAAATTAAAAGAGAAGTATATCATATAA
etfA3         ATGGGTAACGTTTTAGTAGTAATAGAACAAAGAGAAAATGTAAT
SEQ ID NO: 55 TCAAACTGTTTCTTTAGAATTACTAGGAAAGGCTACAGAAATAG
              CAAAAGATTATGATACAAAAGTTTCTGCATTACTTTTAGGTAGT
              AAGGTAGAAGGTTTAATAGATACATTAGCACACTATGGTGCAGA
              TGAGGTAATAGTAGTAGATGATGAAGCTTTAGCAGTGTATACAA
              CTGAACCATATACAAAAGCAGCTTATGAAGCAATAAAAGCAGCT
              GACCCTATAGTTGTATTATTTGGTGCAACTTCAATAGGTAGAGA
              TTTAGCGCCTAGAGTTTCTGCTAGAATACATACAGGTCTTACTG
              CTGACTGTACAGGTCTTGCAGTAGCTGAAGATACAAAATTATTA
              TTAATGACAAGACCTGCCTTTGGTGGAAATATAATGGCAACAAT
              AGTTTGTAAAGATTTCAGACCTCAAATGTCTACAGTTAGACCAG
              GGGTTATGAAGAAAAATGAACCTGATGAAACTAAAGAAGCTGTA
              ATTAACCGTTTCAAGGTAGAATTTAATGATGCTGATAAATTAGT
              TCAAGTTGTACAAGTAATAAAAGAAGCTAAAAAACAAGTTAAAA
              TAGAAGATGCTAAGATATTAGTTTCTGCTGGACGTGGAATGGGT
              GGAAAAGAAAACTTAGACATACTTTATGAATTAGCTGAAATTAT
              AGGTGGAGAAGTTTCTGGTTCTCGTGCCACTATAGATGCAGGTT
              GGTTAGATAAAGCAAGACAAGTTGGTCAAACTGGTAAAACTGTA
              AGACCAGACCTTTATATAGCATGTGGTATATCTGGAGCAATACA
              ACATATAGCTGGTATGGAAGATGCTGAGTTTATAGTTGCTATAA
              ATAAAAATCCAGAAGCTCCAATATTTAAATATGCTGATGTTGGT
              ATAGTTGGAGATGTTCATAAAGTGCTTCCAGAACTTATCAGTCA
              GTTAAGTGTTGCAAAAGAAAAAGGTGAAGTTTTAGCTAACTAA
thiA1         ATGAGAGAAGTAGTAATTGCCAGTGCAGCTAGAACAGCAGTAGG
SEQ ID NO: 56 AAGTTTTGGAGGAGCATTTAAATCAGTTTCAGCGGTAGAGTTAG
              GGGTAACAGCAGCTAAAGAAGCTATAAAAAGAGCTAACATAACT
              CCAGATATGATAGATGAATCTCTTTTAGGGGGAGTACTTACAGC
              AGGTCTTGGACAAAATATAGCAAGACAAATAGCATTAGGAGCAG
              GAATACCAGTAGAAAAACCAGCTATGACTATAAATATAGTTTGT
              GGTTCTGGATTAAGATCTGTTTCAATGGCATCTCAACTTATAGC
              ATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAACA
              TGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCA
              AGAATGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGG
              ATTATCAGACATATTTAATAACTATCACATGGGTATTACTGCTG
              AAAACATAGCAGAGCAATGGAATATAACTAGAGAAGAACAAGAT
              GAATTAGCTCTTGCAAGTCAAAATAAAGCTGAAAAAGCTCAAGC
              TGAAGGAAAATTTGATGAAGAAATAGTTCCTGTTGTTATAAAAG
              GAAGAAAAGGTGACACTGTAGTAGATAAAGATGAATATATTAAG
              CCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTGCATT
              TAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAA
              ATGATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCT
              GAAGAACTAGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGG
              AACAGCTGGTGTTGACCCTAAAATAATGGGATATGGACCAGTTC
              CAGCAACTAAAAAAGCTTTAGAAGCTGCTAATATGACTATTGAA
              GATATAGATTTAGTTGAAGCTAATGAGGCATTTGCTGCCCAATC
              TGTAGCTGTAATAAGAGACTTAAATATAGATATGAATAAAGTTA
              ATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATAGGATGC
              TCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG
              AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTG
              GAATGGGAACTACTTTAATAGTTAAGAGATAG
hbd           ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTAT
SEQ ID NO: 57 TGTACAAACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGA
              GTAGAACTCAAGGTGCTATAGATAAATGTTTAGCTTTATTAGAT
              AAAAATTTAACTAAGTTAGTTACTAAGGGAAAAATGGATGAAGC
              TACAAAAGCAGAAATATTAAGTCATGTTAGTTCAACTACTAATT
              ATGAAGATTTAAAAGATATGGATTTAATAATAGAAGCATCTGTA
              GAAGACATGAATATAAAGAAAGATGTTTTCAAGTTACTAGATGA
              ATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATCAT
              TATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAA
              GTTATAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATT
              AGTTGAAGTTATAAGTGGTCAGTTAACATCAAAAGTTACTTTTG
              ATACAGTATTTGAATTATCTAAGAGTATCAATAAAGTACCAGTA
              GATGTATCTGAATCTCCTGGATTTGTAGTAAATAGAATACTTAT
              ACCTATGATAAATGAAGCTGTTGGTATATATGCAGATGGTGTTG
              CAAGTAAAGAAGAAATAGATGAAGCTATGAAATTAGGAGCAAAC
              CATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGATTAGA
              TGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAG
              ATACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGA
              GCTAATCAATTAGGAAGAAAAACTAAGATAGGATTCTATGATTA
              TAATAAATAA
crt2          ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGA
SEQ ID NO: 58 AGTAGATGGAAATATATGTACAGTGAAAATGAATAGACCTAAAG
              CCCTTAATGCAATAAATTCAAAGACTTTAGAAGAACTTTATGAA
              GTATTTGTAGATATTAATAATGATGAAACTATTGATGTTGTAAT
              ATTGACAGGGGAAGGAAAGGCATTTGTAGCTGGAGCAGATATTG
              CATACATGAAAGATTTAGATGCTGTAGCTGCTAAAGATTTTAGT
              ATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAATAGTAAAAA
              AGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGGAT
              GTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAA
              GCTAAATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGG
              ATATGGAGGAACTCAAAGGCTTACAAGATTGGTTGGAATGGCAA
              AAGCAAAAGAATTAATCTTTACAGGTCAAGTTATAAAAGCTGAT
              GAAGCTGAAAAAATAGGGCTAGTAAATAGAGTCGTTGAGCCAGA
              CATTTTAATAGAAGAAGTTGAGAAATTAGCTAAGATAATAGCTA
              AAAATGCTCAGCTTGCAGTTAGATACTCTAAAGAAGCAATACAA
              CTTGGTGCTCAAACTGATATAAATACTGGAATAGATATAGAATC
              TAATTTATTTGGTCTTTGTTTTTCAACTAAAGACCAAAAAGAAG
              GAATGTCAGCTTTCGTTGAAAAGAGAGAAGCTAACTTTATAAAA
              GGGTAA
pbt           ATGAGAAGTTTTGAAGAAGTAATTAAGTTTGCAAAAGAAAGAGG
SEQ ID NO: 59 ACCTAAAACTATATCAGTAGCATGTTGCCAAGATAAAGAAGTTT
              TAATGGCAGTTGAAATGGCTAGAAAAGAAAAAATAGCAAATGCC
              ATTTTAGTAGGAGATATAGAAAAGACTAAAGAAATTGCAAAAAG
              CATAGACATGGATATCGAAAATTATGAACTGATAGATATAAAAG
              ATTTAGCAGAAGCATCTCTAAAATCTGTTGAATTAGTTTCACAA
              GGAAAAGCCGACATGGTAATGAAAGGCTTAGTAGACACATCAAT
              AATACTAAAAGCAGTTTTAAATAAAGAAGTAGGTCTTAGAACTG
              GAAATGTATTAAGTCACGTAGCAGTATTTGATGTAGAGGGATAT
              GATAGATTATTTTTCGTAACTGACGCAGCTATGAACTTAGCTCC
              TGATACAAATACTAAAAAGCAAATCATAGAAAATGCTTGCACAG
              TAGCACATTCATTAGATATAAGTGAACCAAAAGTTGCTGCAATA
              TGCGCAAAAGAAAAAGTAAATCCAAAAATGAAAGATACAGTTGA
              AGCTAAAGAACTAGAAGAAATGTATGAAAGAGGAGAAATCAAAG
              GTTGTATGGTTGGTGGGCCTTTTGCAATTGATAATGCAGTATCT
              TTAGAAGCAGCTAAACATAAAGGTATAAATCATCCTGTAGCAGG
              ACGAGCTGATATATTATTAGCCCCAGATATTGAAGGTGGTAACA
              TATTATATAAAGCTTTGGTATTCTTCTCAAAATCAAAAAATGCA
              GGAGTTATAGTTGGGGCTAAAGCACCAATAATATTAACTTCTAG
              AGCAGACAGTGAAGAAACTAAACTAAACTCAATAGCTTTAGGTG
              TTTTAATGGCAGCAAAGGCATAA
buk           ATGAGCAAAATATTTAAAATCTTAACAATAAATCCTGGTTCGAC
SEQ ID NO: 60 ATCAACTAAAATAGCTGTATTTGATAATGAGGATTTAGTATTTG
              AAAAAACTTTAAGACATTCTTCAGAAGAAATAGGAAAATATGAG
              AAGGTGTCTGACCAATTTGAATTTCGTAAACAAGTAATAGAAGA
              AGCTCTAAAAGAAGGTGGAGTAAAAACATCTGAATTAGATGCTG
              TAGTAGGTAGAGGAGGACTTCTTAAACCTATAAAAGGTGGTACT
              TATTCAGTAAGTGCTGCTATGATTGAAGATTTAAAAGTGGGAGT
              TTTAGGAGAACACGCTTCAAACCTAGGTGGAATAATAGCAAAAC
              AAATAGGTGAAGAAGTAAATGTTCCTTCATACATAGTAGACCCT
              GTTGTTGTAGATGAATTAGAAGATGTTGCTAGAATTTCTGGTAT
              GCCTGAAATAAGTAGAGCAAGTGTAGTACATGCTTTAAATCAAA
              AGGCAATAGCAAGAAGATATGCTAGAGAAATAAACAAGAAATAT
              GAAGATATAAATCTTATAGTTGCACACATGGGTGGAGGAGTTTC
              TGTTGGAGCTCATAAAAATGGTAAAATAGTAGATGTTGCAAACG
              CATTAGATGGAGAAGGACCTTTCTCTCCAGAAAGAAGTGGTGGA
              CTACCAGTAGGTGCATTAGTAAAAATGTGCTTTAGTGGAAAATA
              TACTCAAGATGAAATTAAAAAGAAAATAAAAGGTAATGGCGGAC
              TAGTTGCATACTTAAACACTAATGATGCTAGAGAAGTTGAAGAA
              AGAATTGAAGCTGGTGATGAAAAAGCTAAATTAGTATATGAAGC
              TATGGCATATCAAATCTCTAAAGAAATAGGAGCTAGTGCTGCAG
              TTCTTAAGGGAGATGTAAAAGCAATATTATTAACTGGTGGAATC
              GCATATTCAAAAATGTTTACAGAAATGATTGCAGATAGAGTTAA
              ATTTATAGCAGATGTAAAAGTTTATCCAGGTGAAGATGAAATGA
              TTGCATTAGCTCAAGGTGGACTTAGAGTTTTAACTGGTGAAGAA
              GAGGCTCAAGTTTATGATAACTAA
ter           ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGC
SEQ ID NO: 61 CCATCCTCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAAT
              ATACCAAGAAACGCATTACCGCAGAAGTCAAAGCTGGCGCAAAA
              GCTCCAAAAAACGTTCTGGTGCTTGGCTGCTCAAATGGTTACGG
              CCTGGCGAGCCGCATTACTGCTGCGTTCGGATACGGGGCTGCGA
              CCATCGGCGTGTCCTTTGAAAAAGCGGGTTCAGAAACCAAATAT
              GGTACACCGGGATGGTACAATAATTTGGCATTTGATGAAGCGGC
              AAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGCGT
              TTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAA
              AAAGGTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCC
              AGTACGTACTGATCCTGATACAGGTATCATGCACAAAAGCGTTT
              TGAAACCCTTTGGAAAAACGTTCACAGGCAAAACAGTAGATCCG
              TTTACTGGCGAGCTGAAGGAAATCTCCGCGGAACCAGCAAATGA
              CGAGGAAGCAGCCGCCACTGTTAAAGTTATGGGGGGTGAAGATT
              GGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGGCCTCTTAGAA
              GAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTGAAGC
              TACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAG
              AACACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCG
              TCAATCCGTGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCG
              CGCAAGCGCCGTAATCCCGGTAATCCCTCTGTATCTCGCCAGCT
              TGTTCAAAGTAATGAAAGAGAAGGGCAATCATGAAGGTTGTATT
              GAACAGATCACGCGTCTGTACGCCGAGCGCCTGTACCGTAAAGA
              TGGTACAATTCCAGTTGATGAGGAAAATCGCATTCGCATTGATG
              ATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCCGCGTTG
              ATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT
              AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATG
              TAGAAGGTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGT
              ATCTGA
tesB          ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA
SEQ ID NO: 15 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG
              GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG
              TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC
              GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA
              TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC
              GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA
              TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC
              AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG
              GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT
              GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC
              CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA
              CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA
              CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA
              ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC
              GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT
              CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG
              AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG
              TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA
              AGGGGTGATGCGTAATCACAATTAA

Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 7.

TABLE 7
Exemplary Polypeptide Sequences for Butyrate Production
Description   Sequence
Bcd2          MDLNSKKYQMLKELYVSFAENEVKPLATELDEEERF
SEQ ID NO: 62 PYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIMAV
              EELSRVCGTTGVILSAHTSLGSWPIYQYGNEEQKQK
              FLRPLASGEKLGAFGLTEPNAGTDASGQQTTAVLDG
              DEYILNGSKIFITNAIAGDIYVVMAMTDKSKGNKGI
              SAFIVEKGTPGFSFGVKEKKMGIRGSATSELIFEDC
              RIPKENLLGKEGQGFKIAMSTLDGGRIGIAAQALGL
              AQGALDETVKYVKERVQFGRPLSKFQNTQFQLADME
              VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFAAE
              TAMEVTTKAVQLHGGYGYTRDYPVERMMRDAKITEI
              YEGTSEVQRMVISGKLLK
etfB3         MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINP
SEQ ID NO: 63 DDKAGLEEAIKLKEEMGAHVTVITMGPPQADMALKE
              ALAMGADRGILLTDRAFAGADTWATSSALAGALKNI
              DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAE
              EIKTEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDM
              NTPRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLG
              LKGSPTSVFKSFTKSVKPAGTIYNEDAKTSAGIIID
              KLKEKYII
etfA3         MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK
SEQ ID NO: 64 VSALLLGSKVEGLIDTLAHYGADEVIVVDDEALAVY
              TTEPYTKAAYEAIKAADPIVVLFGATSIGRDLAPRV
              SARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIM
              ATIVCKDFRPQMSTVRPGVMKKNEPDETKEAVINRF
              KVEFNDADKLVQVVQVIKEAKKQVKIEDAKILVSAG
              RGMGGKENLDILYELAEIIGGEVSGSRATIDAGWLD
              KARQVGQTGKTVRPDLYIACGISGAIQHIAGMEDAE
              FIVAINKNPEAPIFKYADVGIVGDVHKVLPELISQL
              SVAKEKGEVLAN
Ter           MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT
SEQ ID NO: 65 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYGA
              ATIGVSFEKAGSETKYGTPGWYNNLAFDEAAKREGL
              YSVTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL
              ASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGE
              LKEISAEPANDEEAAATVKVMGGEDWERWIKQLSKE
              GLLEEGCITLAYSYIGPEATQALYRKGTIGKAKEHL
              EATAHRLNKENPSIRAFVSVNKGLVTRASAVIPVIP
              LYLASLFKVMKEKGNHEGCIEQITRLYAERLYRKDG
              TIPVDEENRIRIDDWELEEDVQKAVSALMEKVTGEN
              AESLTDLAGYRHDFLASNGFDVEGINYEAEVERFDR
              I
ThiA          MREVVIASAARTAVGSFGGAFKSVSAVELGVTAAKE
SEQ ID NO: 66 AIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG
              AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDAD
              IMLVGGAENMSMSPYLVPSARYGARMGDAAFVDSMI
              KDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL
              ASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDKD
              EYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGINDG
              AAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKIM
              GYGPVPATKKALEAANMTIEDIDLVEANEAFAAQSV
              AVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILTT
              LLYEMKRRDAKTGLATLCIGGGMGTTLIVKR
Hbd           MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI
SEQ ID NO: 67 DKCLALLDKNLTKLVTKGKMDEATKAEILSHVSSTT
              NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED
              TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPM
              MKLVEVISGQLTSKVTFDTVFELSKSINKVPVDVSE
              SPGFVVNRILIPMINEAVGIYADGVASKEEIDEAMK
              LGANHPMGPLALGDLIGLDVVLAIMNVLYTEFGDTK
              YRPHPLLAKMVRANQLGRKTKIGFYDYNK
Crt2          MSTSDVKVYENVAVEVDGNICTVKMNRPKALNAINS
SEQ ID NO: 68 KTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD
              IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA
              VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI
              TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK
              IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSK
              EAIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMS
              AFVEKREANFIKG
Pbt           MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA
SEQ ID NO: 69 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDI
              KDLAEASLKSVELVSQGKADMVMKGLVDTSIILKAV
              LNKEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMN
              LAPDTNIKKQIIENACTVAHSLDISEPKVAAICAKE
              KVNPKMKDTVEAKELEEMYERGEIKGCMVGGPFAID
              NAVSLEAAKHKGINHPVAGRADILLAPDIEGGNILY
              KALVFFSKSKNAGVIVGAKAPIILTSRADSEETKLN
              SIALGVLMAAKA
Buk           MSKIFKILTINPGSTSTKIAVEDNEDLVFEKTLRHS
SEQ ID NO: 70 SEEIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELD
              AVVGRGGLLKPIKGGTYSVSAAMIEDLKVGVLGEHA
              SNLGGIIAKQIGEEVNVPSYIVDPVVVDELEDVARI
              SGMPEISRASVVHALNQKAIARRYAREINKKYEDIN
              LIVAHMGGGVSVGAHKNGKIVDVANALDGEGPFSPE
              RSGGLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVA
              YLNTNDAREVEERIEAGDEKAKLVYEAMAYQISKEI
              GASAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKF
              IADVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN
TesB          MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFG
SEQ ID NO: 41 GQVVGQALYAAKETVPEERLVHSFHSYFLRPGDSKK
              PIIYDVETLRDGNSFSARRVAAIQNGKPIFYMTASF
              QAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLL
              PPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQV
              WIRANGSVPDDLRVHQYLLGYASDLNFLPVALQPHG
              IGFLEPGIQIATIDHSMWFHRPFNLNEWLLYSVEST
              SASSARGFVRGEFYTQDGVLVASTVEGVMRNHN*

The gene products of the bcd2, etfA3, and etfB3 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. In some embodiments, because the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen-limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut. It has been shown that a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose..

In some embodiments, the genetically engineered bacteria of the invention comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd2, etfB3, and effA3, e.g., from Peptoclostridium difficile; and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd2, etfB3, effA3, thiA1, hbd, and crt2, e.g., from Peptoclostridium difficile, and tesB from E. Coli and produce butyrate in low-oxygen conditions, in the presence of molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In one embodiment, the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E. Coli , and produce butyrate in low-oxygen conditions,in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with hunger, appetite, craving, obesity, metablic syndrome, insulin resistance, liver damage, or other condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis.

In some embodiments, the local production of butyrate protects against diet-induced obesity (Lin et al., 2012). In some embodiments, the local production of butyrate protects against diet-induced obesity without causing decreased food intake (Lin et al., 2012). In some embodiments, local butyrate production reduces gut inflammation, a symptom of metabolic disease.

In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. Accordingly, in one embodiment, the bcd2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 53. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 53.

In one embodiment, the etfB3 gene has at least about 80% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the e03 gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. Accordingly, in one embodiment, the e033 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene comprises the sequence of SEQ ID NO: 54. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 54.

In one embodiment, the etfA3 gene has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. Accordingly, in one embodiment, the e03 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 55. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 55.

In one embodiment, the thiA1 gene has at least about 80% identity with SEQ ID NO: 56. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. Accordingly, in one embodiment, the thiA1 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene comprises the sequence of SEQ ID NO: 56. In yet another embodiment the thiAl gene consists of the sequence of SEQ ID NO: 56.

In one embodiment, the hbd gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. Accordingly, in one embodiment, the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 57.

In one embodiment, the crt2 gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 58.

In one embodiment, the pbt gene has at least about 80% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 59.

In one embodiment, the buk gene has at least about 80% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. Accordingly, in one embodiment, the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 60.

In one embodiment, the ter gene has at least about 80% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. Accordingly, in one embodiment, the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 61.

In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15.

In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.

In some embodiments, one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene. The butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.

In some embodiments, the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production. In some embodiments, the local production of butyrate reduces food intake and ameliorates metabolic disease (Lin et al., 2012). In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the butyrate gene cassette is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

The butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the butyrate gene cassette is expressed on a low- copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate.

Acetate

In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and produce acetate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale et al., 2008). In some embodiments, the genetically engineered bacteria of the invention comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli. In some embodiments, the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and/or Thermoacetogenium. The genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.

In some embodiments, the genetically engineered bacteria produce acetate and butyrate, as described herein (see, e.g., FIG. 13 and FIG. 14). GLP-1

In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-1 or proglucagon. GLP-1 and several other insulin and satiety regulating peptides result from cleaved of preproglucagon. Preproglucagon is proteolytically cleaved in a tissue-specific manner. Post-translational processing in the gut and brain by prohormone convertases results in the secretion of GLP-1 and GLP-2, while the glucagon sequence remains in a larger peptide, glicentin or glicentin-related pancreatic peptide (GRPP) and oxyntomodulin. Glucagon-like peptide 1 (GLP-1) is produced by intestinal cells, e.g., ileal L cells, and is capable of stimulating insulin secretion and the differentiation of insulin-secreting cells and inhibiting glucagon secretion. GLP-1 is capable of restoring glucose sensitivity and increasing satiety.

Glucagon-like peptide 1 (GLP-1) is also used to treat those suffering from non-alcoholic steatohepatitis by reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition. Glucagon-like peptide 1 is well known to those of skill in the art. For example, glucagon-like peptide 1 has been used to stimulate insulin secretion in the treatment of type-two diabetes and non-alcoholic steatohepatitis (NASH). See, for example, Armstrong, et al., J. of Hepatology, 64:399-408 (2016); Bernsmeier, et al., PLOS One, 9(1): e87488 (2014); Kjems, et al., Diabetes, 52:380-386 (2003); Knudsen et al., J. Med. Chem., 43:1664-1669 (2000); MacDonald, et al., Diabetes, 51(supp. 3):5434-5442 (2002); Werner, et al., Regulatory Peptides, 164:58-34 (2010); Drucker and Nauck, Lancet, 368:1696-1705 (2006); Jiminez-Solem, et al., Cur. Opinion in Mol. Therap., 12(6):760-797 (2010); Schnabel, et al., Vasc. Health and Risk Mgmt., 2(1):69-77 (2006); and WO1995/017510, the entire contents of each of which are expressly incorporated herein by reference.

Proteolytic cleavage of proglucagon produces GLP-1 and GLP-2. GLP-1 adminstration has therapeutic potential in treating type 2 diabetes (Gallwitz et al., 2000). The genetically engineered bacteria may comprise any suitable gene encoding GLP-1 or proglucagon, e.g., human GLP-1 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-1 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-1 analog (see, e.g., Gallwitz et al., 2000). In some embodiments, the gene encoding GLP-1 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-1 production, and/or increase metabolic disease attenuation potency. In some embodiments, the local production of GLP-1 induces insulin secretion and/or differentiation of insulin-secreting cells. In some embodiments, the local production of GLP-1 produces satiety in a subject and ameliorates obesity. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-1 or proglucagon in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

TABLE 8
GLP-1 Polynucleotide Sequences
Description              Sequence
GLP-1 (1-37), with       ATGGACGAGTTCGAACGCCACG
initiation met codon;    CGGAGGGAACTTTCACTTCTGA
codon optimized for      TGTTTCTAGCTATTTGGAGGGC
expression in E. coli.   CAGGCTGCGAAAGAGTTTATTG
SEQ ID NO: 71            CTTGGCTGGTTAAAGGTCGTGG
                         TTAA
GLP1 (1-37) codon        GACGAGTTCGAACGCCACGCGG
optimized for expression AGGGAACTTTCACTTCTGATGT
in E. coli.              TTCTAGCTATTTGGAGGGCCAG
SEQ ID NO: 72            GCTGCGAAAGAGTTTATTGCTT
                         GGCTGGTTAAAGGTCGTGGTTA
                         A

TABLE 9
GLP-1 Polypeptide Sequences
Description                     Sequence
GLP-1 (1-37)                    HDEFERHAEGTFTSDVSSYLEGQAAKEFIAW
SEQ ID NO: 73                   LVKGRG
GLP-1 (1-37) H→M substitution   MDEFERHAEGTFTSDVSSYLEGQAAKEFIAW
SEQ ID NO: 74                   LVKGRG
GLP-1-(7-37)                    HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
SEQ ID NO: 75
GLP-1-(7-36)NH2                 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
SEQ ID NO: 76
glucagon preproprotein          MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSR
(NP_002045.1) 1-20 is signal    SFSASQADPLSDPDQMNEDKRHSQGTFTSDY
peptide                         SKYLDSRRAQDFVQWLMNTKRNRNNIAKRHD
SEQ ID NO: 77                   EFERHAEGTFTSDVSSYLEGQAAKEFIAWLV
                                KGRGRRDFPEEVAIVEELGRRHADGSFSDEM
                                NTILDNLAARDFINWLIQTKITDRK
Proglucagon (Signal peptide 1-  RSLQDTEEKSRSFSASQADPLSDPDQMNEDK
20; Giucagon-like. peptide 1    RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK
(92-128); Glucagon-like peptide RNRNNIAKRHDEFERHAEGTFTSDVSSYLEG
2 146-178                       QAAKEFIAWLVKGRGRRDFPEEVAIVEELGR
SEQ ID NO: 78                   RHADGSFSDEMNTILDNLAARDFINWLIQTK
                                ITDRK
Glucagon                        HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
SEQ ID NO: 79
Glicentin                       RSLQDTEEKSRSFSASQADPLSDPDQMNEDK
SEQ ID NO: 80                   RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK
                                RNRNNIA
Glicentin related peptide       RSLQDTEEKSRSFSASQADPLSDPDQMNED
SEQ ID NO: 81
Oxyntomodulin                   HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKR
SEQ ID NO: 82                   NRNNIA

The circulating active form of GLP-1 is GLP-1(7-37), which has a very short biological half-life of the order of just a few minutes in blood. The relatively low stability of GLP-1 (3-5 min) has significantly limited its clinical utility because of the rapid degradation catalyzed by the enzyme dipeptidyl peptidase IV (DPP-IV), but also other enzymes such as neutral endopeptidase (NEP), plasma kallikrein or plastnin. One strategy to prolong in vivo half-life is stabilization towards degradation by DPPIV, which preferably cleaves N-terminal Xaa-Pro or Xaa-Ala dipeptide sequences. Alteration of that N-terminal sequence, especially the second amino acid, has proven to reduce degradation by DPPIV (e.g., reviewed in Lorenz et al., Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity; Bioorganic & Medicinal Chemistry Letters, 23 (14); 4011-4018). In some embodiments, the genetically engineered bacteria comprise a cassette encoding GLP-1 fragment or variant, in which the DPP-IV is mutated, such that it can no longer be cleaved by the enzyme.

GLP-1 is released in a tissue specific manner, though post-translational processing of pre-pro-glucagon, from the neuroendocrine L-cells predominantly in two forms, GLP-1 (7-36) amide, which constitutes approximately 80% of circulating GLP-1, and GLP-1 (7-37) amide. GLP-1 (1-36 amide) is predominantly secreted in the pancreas, whereas GLP-1 (1-37) is secreted in the ileum and hypothalamus.

In addition, full length GLP-1-(1-37) is produced in much smaller amounts. This full-length form of GLP-1(1-37), was previously thought to be inactive, but was found to stimulate rat intestinal epithelial cells to become glucose-responsive insulin-secreting cells, i.e., full length GLP-1 could convert intestinal epithelial progenitors in the small intestine into insulin-producing cells (Suzuki et al., Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells; Proc Natl Acad Sci U S A. 2003 Apr. 29; 100(9): 5034-5039). While the amounts of GLP-1 (1-37) produced endogenously likely are not sufficient for these effects, secretion of large amounts of GLP-1, e.g., by the genetically engineered bacteria, are likely sufficient to alter a balance in the developmental environment of the intestinal epithelia, leading to the induction of insulin-producing cells from intestinal epithelial progenitors. As such, secretion of full-length GLP-1 by the genetically engineered bacteria of the disclosure is a novel therapeutic strategy for the treatment of a number of diseases related to dysregulation of insulin production and/or secretion, including diabetes.

GLP-1 analogs, which exhibit extended stability in serum, have become important in the clinic. Exendin-4, a peptide produced in the salivary glands of the Gila monster (Heloderma suspectum), possesses similar glucose regulatory function to the human GLP-1 peptide. In exendin-4, the second amino acid is a Gly rendering it resistant to DPPIV mediated degradation. Furthermore, the Leu21-Ser39 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure, leading to enhanced helicity and stability of the peptide (see Lorenz et al. for review). Exenatide BID is a synthetic version of exendin-4, represents the first GLP-1 RA approved in 2005 as antidiabetic therapy for the treatment of T2DM. Following the FDA approval of exendin-4, liraglutide and albiglutide, which are long-acting GLP-1 analogs using palmitic acid conjugation and albumin fusion, respectively, were approved. Many other strategies have also been employed to achieve long-acting activity of GLP-1, including dimerization, intra-molecular conjugation, and additional variant positive charged amino acids on the N terminus. Table 10 lists non-limiting examples of GLP-1R agonists. In some embodiments, the genetically engineered bacteria comprise a gene encoding Exenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Liraglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Lixisenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Albiglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Dulaglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Taspoglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Semaglutide.

TABLE 10
Non-limiting examples of GLP-1R agonists
Name and SEQ ID NO	Sequence	Short description
Exenatide	HGEGTFTSDLSKQMEE	Second amino acid is a Gly
SEQ ID NO: 83	EAVRLFIEWLKNGGPS	rendering it resistant to
	SGAPPPS	DPPIV mediated
		degradation. Furthermore,
		the Leu21-Ser39 span of
		exendin-4 forms a compact
		tertiary fold (the Trp-cage)
		which shields the side chain
		of Trp25 from solvent
		exposure, leading to
		enhanced helicity and
		stability of the peptide
Liraglutide	HAEGTFTSDVSSYLEG	a close structural homolog to
SEQ ID NO: 84	QAAKEEFIIAWLVKGR	GLP-1(7-37) with 97%
	G	sequence identity to the
		native hormone. Lys in
		position 34 is substituted by
		Arg and a palmitic acid is
		conjugated to Lys in position
		26 via a glutamate spacer
Lixisenatide	HGEGTFTSDLSKQMEE	synthetic analog of exendin-
SEQ ID NO: 85	EAVRLFIEWLKNGGPS	4. Compared to exendin-4,
	SGAPPSKKKKKK	six Lys residues have been
		added to the C-terminus (also
		amidated), while one Pro in
		the C-terminal region has
		been deleted.
Albiglutide	HGEGTFTSDVSSYLEG	two copies of GLP-1 are
SEQ ID NO: 86	QAAKEFIAWLVKGRH	fused as tandem repeat to the
	GEGTFTSDVSSYLEGQ	N-terminus of albumin.
	AAKEFIAWLVKGRDA	DPPIV-resistance is
	HKSEVAHRFKDLGEEN	achieved by a single
	FKALVLIAFAQYLQQC	substitution, Ala for Gly, at
	PFEDHVKLVNEVTEFA	the DPPIV cleavage site.
	KTCVADESAENCDKSL
	HTLFGDKLCTVATLRE
	TYGEMADCCAKQEPE
	RNECFLQHKDDNPNLP
	RLVRPEVDVMCTAFH
	DNEETFLKKYLYEIAR
	RHPYFYAPELLFFAKR
	YKAAFTECCQAADKA
	ACLLPKLDELRDEGKA
	SSAKQRLKCASLQKFG
	ERAFKAWAVARLSQR
	FPKAEFAEVSKLVTDL
	TKVHTECCHGDLLECA
	DDRADLAKYICENQDS
	ISSKLKECCEKPLLEKS
	HCIAEVENDEMPADLP
	SLAADFVESKDVCKN
	YAEAKDVFLGMFLYE
	YARRHPDYSVVLLLRL
	AKTYETTLEKCCAAA
	DPHECYAKVFDEFKPL
	VEEPQNLIKQNCELFE
	QLGEYKFQNALLVRY
	TKKVPQVSTPTLVEVS
	RNLGKVGSKCCKHPE
	AKRMPCAEDYLSVVL
	NQLCVLHEKTPVSDRV
	TKCCTESLVNRRPCFS
	ALEVDETYVPKEFNAE
	TFTFHADICTLSEKERQ
	IKKQTALVELVKHKPK
	ATKEQLKAVMDDFAA
	FVEKCCKADDKETCFA
	EEGKKLVAASQAALG
	L
Dulaglutide	HGEGTFTSDVSSYLEE	A recombinant fusion
SEQ ID NO: 87	QAAKEFIAWLVKGGG	protein, which consists of
	GGGGSGGGGSGGGGS	two GLP-1 peptides
	AESKYGPPCPPCPAPE	covalently linked by a small
	AAGGPSVFLFPPKPKD	peptide [tetraglycyl-1-
	TLMISRTPEVTCVVVD	seryltetraglycyl-1-
	VSQEDPEVQFNWYVD	seryltetraglycyl-1-seryl-1-
	GVEVHNAKTKPREEQF	alanyl] to a human IgG4-Fc
	NSTYRVVSVLTVLHQD	heavy chain variant.
	WLNGKEYKCKVSNKG	Compared to natural GLP-1,
	LPSSIEKTISKAKGQPR	the GLP-1 moieties contain
	EPQVYTLPPSQEEMTK	amino acid substitutions
	NQVSLTCLVKGFYPSD	(Ala8→Gly, Gly26→Glu,
	IAVEWESNGQPENNYK	Arg36→Gly) to ensure
	TTPPVLDSDGSFFLYSR	protection from DPPIV
	LTVDKSRWQEGNVFS	cleavage as well as
	CSVMHEALHNHYTQK	maintenance of the potency
	SLSLSLG	of the construct.
Taspoglutide	His-Aib-Glu-Gly-Thr-	a close analog of natural
SEQ ID NO: 88	Phe-Thr-Ser-Asp-Val-Ser-	GLP-1(7-36) in which the
	Ser-Tyr-Leu-Gly-Gly-	unnatural amino acid
	Gln-Ala-Ala-Lys-Glu-	aminoisobutyric acid (Aib)
	Phe-Ile-Ala-Trp-Leu-Val-	has been introduced in
	Lys-Aib-Arg-NH2	position 8 and 35 in order to
		avoid degradation by DITIV,
		hut also by other serine
		proteases such as plasma
		kallikrein and plasmin.
Semaglutide	MAGAPGPLRLALLLLG
SEQ ID NO: 89	MVGRAGPRPQGATVS
	LWETVQKWREYRRQC
	QRSLTEDPPPATDLFC
	NRTFDEYACWPDGEP
	GSFVNVSCPWYLPWA
	SSVPQGHVYRFCTAEG
	LWLQKDNSSLPWRDL
	SECEESKRGERSSPEEQ
	LLFLYIIYTVGYALSFS
	ALVIASAILLGFRHLHC
	TRNYIHLNLFASFILRA
	LSVFIKDAALKWMYST
	AAQQHQWDGLLSYQD
	SLSCRLVFLLMQYCVA
	ANYYWLLVEGVYLYT
	LLAFSVLSEQWIFRLY
	VSIGWGVPLLFVVPWG
	IVKYLYEDEGCWTRNS
	NMNYWLIIRLPILFAIG
	VNFLIFVRVICIVVSKL
	KANLMCKTDIKCRLA
	KSTLTLIPLLGTHEVIF
	AFVMDEHARGTLRFIK
	LFTELSFTSFQGLMVAI
	LYCFVNNEVQLEFRKS
	WERWRLEHLHIQRDSS
	MKPLKCPTSSLSSGAT
	AGSSMYTATCQASCS

In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 stimulates the rate of insulin secretion in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 inhibits and lowers plasma glucose produced in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of lipotoxic metabolites in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the degree of pro-inflammatory substrate in the body. In one embodiment, GLP-1 decreases the level of insulin resistance (IR) in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of hepatic lipid deposition in the body. Methods for measuring the insulin secretion rates and glucose levels are well known to one of ordinary skill in the art. For example, blood samples taken periodically, and standard statistical analysis methods may be used to determine the insulin secretion rates and plasma glucose levels in a subject.

GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having liver disease, such as NASH. Specifically, when GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells of the disclosure, the expressed GLP-1 and/or a GLP-1R agonist of Table 10 will reduce the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition in the subject.

GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having type two diabetes, obesity, and/or metabolic syndrome, or metabolic syndrome related disorders, including cardiovascular disorders, and obesity in a subject.

In one embodiment, the bacterial cell comprises one or more genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a glucagon-like peptide 1 operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a glucagon-like peptide 1. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a glucagon-like peptide 1.

Multiple distinct embodiments of GLP-1 and/or a GLP-1R agonist of Table 10 are known in the art. In some embodiments, the glucagon-like peptide 1 is encoded by a gene derived from a bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a non-bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a eukaryotic species, e.g. homo sapiens. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed in an organism of the genus or species that includes, but is not limited to, Lactobacillus spp., such as Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus brevis, or Lactobacillus gasseri; Bifidobacterium spp., such as Bifidobacterium longum; Bacillus spp., such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus; and Streptomyces spp., such as Streptomyces lividans.

In one embodiment, the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in Escherichia coli. In another embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in Lactococcus. When the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells, the bacterial cells express more GLP-1 and/or a GLP-1R agonist of Table 10 than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 may be used to express more GLP-1 and/or a GLP-1R agonist of Table 10 to treat liver disease, such as nonalcoholic steatohepatitis, type two diabetes, metabolic syndrome, and metabolic syndrome related disorders, including cardiovascular disorders and obesity in a subject.

Assays for testing the activity of a GLP-1 and/or a GLP-1R agonist of Table 10 or a glucagon-like peptide 1 receptor are well known to one of ordinary skill in the art. For example, glucose and insulin levels can be assessed by drawing plasma samples from subjects previously administered intravenous infusions of the glucagon- like peptide 1 as described in Kjems, et al., Diabetes, 52:380-386 (2003), the entire contents of which are expressly incorporated herein by reference. Briefly, plasma samples from a subject are treated with heparin and sodium fluoride, centrifuged, and plasma glucose levels measured by a glucose oxidase technique. Likewise, the plasma insulin concentrations are measured by a two-site insulin enzyme linked immunosorbent method. Alternatively, baby hamster kidney cells can be used to assay structure-activity relationships of glucagon-like peptide 1 derivatives (see, for example, Knudsen et al., J. Med. Chem., 43:1664-1669 (2000), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10 comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.

In some embodiments, the gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the gene encoding the glucagon-like peptide 1 is from Homo sapiens. In one embodiment, the gene encoding the glucagon-like peptide 1 is from Lactobacillus spp. In one embodiment, the Lacotbacillus spp. is Lactobacillus plantarum WCFS1, Lactobacillus plantarum 80, Lactobacillus johnsonii NCC533, Lactobacillus johnsonii 100-100, Lactobacillus acidophilus NCFM ATCC700396, Lactobacillus brevis ATCC 367, Lactobacillus gasseri ATCC 33323, or Lactobacillus acidophilus. In another embodiment, the gene encoding the glucagon-like peptide 1 is from a Bifidobacterium spp. In one embodiment, the Bifidobacterium spp. is Bifidobacterium longum NCC2705, Bifidobacterium longum DJ010A, Bifidobacterium longum BB536, or Bifidobacterium longum SBT2928. In another embodiment, the gene encoding the glucagon-like peptide 1 is from Bacillus spp. In one embodiment, the Bacillus spp is Bacillus subtilis, or Bacillus licheniformis, or Bacillus lentus, or Bacillus brevis, or Bacillus stearothermophilus, or Bacillus alkalophilus, or Bacillus amyloliquefaciens, or Bacillus coagulans, or Bacillus circulans, or Bacillus lautus. In another embodiment, the gene encoding the glucagon-like peptide 1 is from Streptomyces spp. In one embodiment, the Streptomyces spp. is Streptomyces lividans. Other genes encoding glucagon-like peptide 1 are well-known to one of ordinary skill in the art and described in, for example, MacDonald, et al., Diabetes, 51(supp. 3):S434-S442 (2002) and WO1995/017510.

In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 80% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 85% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 90% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 95% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. Accordingly, in one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding SEQ ID NO:40. In another embodiment, the gene encoding the glucagon-like peptide 1 comprises a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In yet another embodiment the gene encoding the glucagon-like peptide 1 consists of a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72.

In one embodiment, the gene encoding the glucagon-like peptide 1 is directly operably linked to a first promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is indirectly operably linked to a first promoter. In one embodiment, the gene encoding the glucagon-like peptide 1 is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the glucagon-like peptide 1 is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. Inducible promoters are described in more detail infra.

The gene encoding the glucagon-like peptide 1 may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located in the chromosome of the bacterial cell.

In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a low-copy plasmid. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the glucagon-like peptide 1, thereby reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition prevalent to those suffering from non-alcoholic steatohepatitis.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 73. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37) H->M substitution), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 74. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 75. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-36), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 76.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding glucagon preproprotein (NP_002045.1), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Proglucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 78. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 79. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 80 In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin related peptide), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 81. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Oxyntomodulin. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 82.

In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 73 through SEQ ID NO: 82.

In embodiments, the pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs described herein, and functional variants or fragments thereof are secreted. In some embodiments, the genetically engineered bacteria comprise one or more cassettes encoding pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs, and/or functional variants or fragments and a secretion gene cassette and/or mutations generating a leaky phenotype. In some embodiments, a flagellar type III secretion pathway is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Type V Autotransporter Secretion System is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Hemolysin-based Secretion System is used to secrete the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein.. In alternate embodiments, the genetically engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein further comprise a non-native single membrane-spanning secretion system. As described herein. In some embodiments, the engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. have one or more deleted or mutated membrane genes to generate a leaky phenotype as described herein.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Exenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 83.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Liraglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 84. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Lixisenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 85. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Albiglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 86. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Dulaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 87. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Taspoglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding

SEQ ID NO: 88. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Semaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 89.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 83 through SEQ ID NO: 89.

IL-22

In some embodiments, the genetically engineered bacteria are capable of producing IL-22. Interleukin 22 (IL-22) cytokine can be produced by dendritic cells, lymphoid tissue inducer-like cells, natural killer cells and expressed on adaptive lymphocytes. Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

As described by Wang et al, (Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes, Nature 514, 237-241 (9 Oct. 2014)) mice which are deficient in IL-22 receptor and are fed a high-fat diet have a propensity to the development of metabolic disorders. Moreover, Wang et al found that administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. These results indicate that IL-22 shows metabolic benefits, from positively affecting insulin sensitivity to the preservation of gut barreier integrity. IL-22 further affects endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues.

Bile Salts

Bile salts (also called conjugated bile acids) are cholesterol derivatives synthesized in the liver which comprise a steroid ring component conjugated with either taurine (taurocholic acid; TCA) or glycine (glycochenodeoxycholic acid; GCDCA). Bile salts act as signaling molecules to regulate systemic endocrine functions, including triglyceride, cholesterol, and glucose homeostasis (Houten et al., EMBO J., 25:1419-1425 (2006) and Watanabe et al., Nature, 439:484-489 (2006)). Specifically, bile acids trigger cellular farnesoid X receptor (FXR)- and G-protein coupled receptor (TGR4)-mediated host responses. Additionally, bile salts have been shown to facilitate lipid absorption and repress bacterial cell growth in the small intestine, thereby influencing both host metabolic pathways and the microflora present in the gut (Jones et al., PNAS, 105(36):13580-13585 (2008) and Ridlon et al., J. Lipid Research, 47(2):241-259 (2006)).

Bile salts are stored in the gallbladder and then subsequently released into the duodenum via the common bile duct. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules, a process referred to as deconjugation, to produce the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. Once in the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA) (Joyce et al., Gut Microbes, 5(5):669-674 (2014); Bhowmik et al., Accepted Article, doi:10.1002/prot.24971 (2015)).

It has been shown that bile salt metabolism is involved in host physiology (Ridlon et al., Current Opinion Gastroenterol., 30(3):332 (2014) and Jones et al., 2008). For example, it is known that the expression of bile salt hydrolase enzymes functionally regulates host lipid metabolism and play a role in cholesterol metabolism and transport, circadian rhythm, gut homeostasis/barrier function, weight gain, adiposity, and possibly gastrointestinal cancers in the host (Joyce et al., PNAS, 111(20):7421-7426 (2014); Zhou and Hylemon, Steroids, 86:62-68, (2014); Mitchell et al., Expert Opinion Biolog. Therapy, 13(5):631-642 (2013); and WO14/198857, the entire contents of each of which are expressly incorporated herein by reference). Specifically, potential effects of bile salt hydrolase-expressing bacteria on cholesterol metabolic pathways have been shown to upregulate the ATP binding cassette A1 (ABCA1), the ATP binding cassette G1 (ABCG1), the ATP binding cassette G5/G8 (ABCGS/G8), cholesterol 7 alpha-hydroxylase (CYP7A1), and liver X receptor (LXR), and to downregulate farnesoid X receptor (FXR), Niemann-Pick C1-like 1 (NPC1L1), and small heterodimer partner (SHP), which impacts cholesterol efflux, plasma HDL-C levels, biliary excretion, cholesterol catabolism, bile acid synthesis, cholesterol levels, and decreased intestinal cholesterol absorption, among other effects (Mitchel et al. (2014) and Zhou and Hylemon (2014)). Additionally, bile salt hydrolase activity has been shown to impact bile detoxification, gastrointestinal persistence, nutrition, membrane alterations, altered digestive functions (lipid malabsorption, weight loss), cholesterol lowering, cancer, and formation of gallstones (see Begley et al., Applied and Environmental Microbiology, 72(3):1729-1738 (2006)). Moreover, a Clostridium scindens bacterium expressing a 7α-dehydroxylase enzyme has been shown to produce resistance to C. difficile infection in hosts (Buffie et al., Nature, 517:205-208 (2015), and bile salt metabolism has been shown to play a role in both regulating the microbiome as well as in cirrhosis (Ridlon et al., Gut Microbes, 4(5):382-387 (2013) and Kakiyama et al., J. Hepatol., 58(5):949-955 (2013)). Thus, a need exists for treatments which address the metabolism of bile salts in subjects in order to treat and prevent diseases and disorders in which bile salts play a role, such as cardiovascular disease, metabolic disease, cirrhosis, gastrointestinal cancer, and C. difficile infection.

As used herein, the term “bile salt” or “conjugated bile acid” refers to a cholesterol derivative that is synthesized in the liver and consists of a steroid ring component that is conjugated with either glycine (glycochenodeoxycholic acid; GCDCA) or taurine (taurocholic acid; TCA). Bile salts are stored in the gallbladder and then subsequently released into the duodenum. Bile salts act as signaling molecules to regulate systemic endocrine functions including triglyceride, cholesterol, and glucose homeostasis, and also facilitate lipid absorption. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules to produce bile acids.

As used herein, the term “bile acid” or “unconjugated bile acid” refers to cholic acid (CA) or chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. In the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA). As used herein, the term “catabolism” refers to the processing, breakdown and/or degradation of a metabolite or a complex molecule, such as tryptophan or a bile salt, into compounds that are non-toxic or which can be utilized by the bacterial cell or can be exported inot the extracellular environment, where these compounds may function as effectors.

In one embodiment, the term “bile salt catabolism” refers to the processing, breakdown, and/or degradation of bile salts into unconjugated bile acid(s). In one embodiment, “abnormal catabolism” refers to any condition(s), disorder(s), disease(s), predisposition(s), and/or genetic mutations(s) that result in increased levels of bile salts. In one embodiment, “abnormal catabolism” refers to an inability and/or decreased capacity of a cell, organ, and/or system to process, degrade, and/or secrete bile salts. In healthy adult humans, 600 mg of bile salts are secreted daily. In one embodiment, said inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts is caused by the decreased endogenous deconjugation of bile salts, e.g., decreased endogenous deconjugation of bile salts into bile acids by the intestinal microbiota in the gut. In one embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts results from a decrease in the number of or activity of intestinal bile salt hydrolase (BSH)-producing microorganisms.

In one embodiment, a “disease associated with bile salts” or a “disorder associated with bile salts” is a disease or disorder involving the abnormal, e.g., increased, levels of bile salts in a subject. Alternatively, a disease or disorder associated with bile salts is a disease or disorder wherein a subject exhibits normal levels of bile salts, but wherein the subject would benefit from decreased levels of bile salts. Bile salts function to solubilize dietary fat and enable its absorption into host circulation, and healthy adult humans secrete about 600 mg of bile salts daily through the stool. Thus, decreasing increased levels of bile salts, or normal levels of bile salts, in a subject would result in less uptake of dietary fat, causing the subject's liver to pull cholesterol from systemic circulation as it attempts to synthesize more. Thus, in one embodiment, a subject having a disease or disorder associated with bile salts secretes about 600 mg of bile salts in their stool daily. In another embodiment, a subject having a disease or disorder associated with bile salts secretes more than 600 mg, 700 mg, 800 mg, 900 mg, or 1 g of bile salts in their stool daily.In one embodiment, a disease or disorder associated with bile salts is a cardiovascular disease. In another embodiment, a disease or disorder associated with bile salts is a metabolic disease. In another embodiment, a disease or disorder associated with bile salts is a liver disease, such as cirrhosis, nonalcoholic steatohepatitis (NASH), or progressive familialintrahepatic cholestasis type 2 (PFIC2).

As used herein, the terms “cardiovascular disease” or “cardiovascular disorder” are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body, and encompasses diseases and conditions including, but not limited to hypercholesterolemia, diabetic dyslipidemia, hypertension, arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease. As used herein, a subject having “hypercholesterolemia” may have a total cholesterol of greater than 4 mmol/L, and a low-density lipoprotein cholesterol (LDL) of greater than 3mmol/L.

As used herein, the term “bile salt hydrolase” enzyme refers to an enzyme involved in the cleavage of the amino acid sidechain of glycol- or tauro-conjugated bile acids to generate unconjugated bile acids (FIG. 2). Bile salt hydrolase (BSH) enzymes are well known to those of skill in the art. For example, bile salt hydrolase activity has been detected in Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Clostridum spp., Bacteroides spp., Methanobrevibacter spp., and Listeria spp. See, for example, Begley et al., Applied and Environmental Microbiology, 72(3):1729-1738 (2006); Jones et al., Proc. Natl. Acad. Sci., 105(36):13580-13585 (2008); Ridlon et al., J. Lipid Res., 47(2):241-259 (2006); and WO2014/198857, the entire contents of each of which are expressly incorporated herein by reference.

Bile Salt Hydrolases

The bacterial cells described herein comprise a heterologous gene encoding a bile salt hydrolase enzyme and are capable of deconjugating bile salts into unconjugated bile acids (see FIG. 27 and FIG. 28).

In one embodiment, the bile salt hydrolase enzyme increases the rate of bile salt catabolism in the cell. In one embodiment, the bile salt hydrolase enzyme decreases the level of bile salts in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of taurocholic acid (TCA) in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of glycochenodeoxycholic acid (GCDCA) in the cell or in the subject. Methods for measuring the rate of bile salt catabolism and the level of bile salts and bile acids are well known to one of ordinary skill in the art. For example, bile salts and acids may be extracted from a sample, and standard LC/MS methods may be used to determine the rate of bile salt catabolism and/or level of bile salts and bile acids.

In another embodiment, the bile salt hydrolase enzyme increases the level of bile acids in the cell or in the subject as compared to the level of bile salts in the cell or in the subject. In another embodiment, the bile salt hydrolase enzyme increases the level of cholic acid (CA) in the cell. In another embodiment, the bile salt hydrolase enzyme increases the level of chenodeoxycholic acid (CDCA) in the cell.

Enzymes involved in the catabolism of bile salts may be expressed or modified in the bacteria of the disclosure in order to enhance catabolism of bile salts. Specifically, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts into unconjugated bile acids when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts, such as TCA or GCDCA, into CA and CDCA when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme can catabolize bile salts to treat disorders associated with bile salts, including cardiovascular diseases, metabolic diseases, liver disease, such as cirrhosis or NASH, gastrointestinal cancers, and C. difficile infection.

In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a bile salt hydrolase enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a bile salt hydrolase enzyme, as well as at least one copy of a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a bile salt hydrolase enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a bile salt hydrolase enzyme.

Multiple distinct bile salt hydrolase enzymes are known in the art. In some embodiments, bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a non-bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene derived from a eukaryotic species, e.g., fungi. In one embodiment, the gene encoding the bile salt hydrolase enzyme is derived from an organism of the genus or species that includes, but is not limited to, Lactobacillus spp., such as Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus brevis, or Lactobacillus gasseri; Bifidobacterium spp., such as Bifidobacterium longum, Bifidobacterium bifidum, or Bifidobacterium adolescentis; Bacteroides spp., such as Bacteroides fragilis or Bacteroides vlugatus; Clostridium spp., such as Clostridium perfringens; Listeria spp., such as Listeria monocytogenes, Enterococcus spp., such as Enterococcus faecium or Enterococcus faecalis; Brucella spp., such as Brucella abortus; Methanobrevibacter spp., such as Methanobrevibacter smithii, Staphylococcus spp., such as Staphylococcus aureus, Mycobacterium spp., such as Mycobacterium tuberculosis; Salmonella spp., such as Salmonella enterica; Listeria spp., such as Listeria monocytogenes.

In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Lactococcus. When the gene encoding the bile salt hydrolase enzyme is expressed in the recombinant bacterial cells, the bacterial cells catabolize more bile salt than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme may be used to catabolize excess bile salts to treat a disorder associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH.

The present disclosure further comprises genes encoding functional fragments of a bile salt hydrolase enzyme or functional variants of a bile salt hydrolase enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a bile salt hydrolase enzyme relates to an element having qualitative biological activity in common with the wild-type bile salt hydrolase enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated bile salt hydrolase enzyme is one which retains essentially the same ability to catabolize bile salts as the bile salt hydrolase enzyme from which the functional fragment or functional variant was derived. For example, a polypeptide having bile salt hydrolase enzyme activity may be truncated at the N-terminus or C-terminus and the retention of bile salt hydrolase enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional variant. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional fragment.

Assays for testing the activity of a bile salt hydrolase enzyme, a bile salt hydrolase enzyme functional variant, or a bile salt hydrolase enzyme functional fragment are well known to one of ordinary skill in the art. For example, bile salt catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous bile salt hydrolase enzyme activity. Bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner, Applied Environ. Microbiol., 55:11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992), the entire contents of each of which are expressly incorporated herein by reference. Briefly, bacterial cultures that are grown overnight can be spotted onto LB bile agar supplemented with either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile. BSH activity can be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al., PNAS, 105(36):13580-13585 (2008), the entire contents of which are expressly incorporated herein by reference). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3):186-202 (2012), the entire contents of which are expressly incorporated herein by reference. Alternatively, a mouse model can be used to assay bile salt and bile acid signatures in vivo (see, for example, Joyce et al., PNAS, 111(20):7421-7426 (2014), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a bile salt hydrolase enzyme comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.

In some embodiments, the gene encoding a bile salt hydrolase enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the bile salt hydrolase enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Lactobacillus spp. In one embodiment, the Lacotbacillus spp. is Lactobacillus plantarum WCFS1, Lactobacillus plantarum 80, Lactobacillus johnsonii NCC533, Lactobacillus johnsonii 100-100, Lactobacillus acidophilus NCFM ATCC700396, Lactobacillus brevis ATCC 367, Lactobacillus gasseri ATCC 33323, or Lactobacillus acidophilus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from a Bifidobacterium spp. In one embodiment, the Bifidobacterium spp. is Bifidobacterium longum NCC2705, Bifidobacterium longum DJO10A, Bifidobacterium longum BB536, Bifidobacterium longum SBT2928, Bifidobacterium bifidum ATCC 11863, or Bifidobacterium adolescentis. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Bacteroides spp. In one embodiment, the Bacteroides spp. is Bacteroides fragilis or Bacteroides vlugatus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Clostridium spp. In one embodiment, the Clostridum spp. is Clostridum perfringens MCV 185 or Clostridum perfringens 13. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Listeria spp. In one embodiment, the Listeria spp. is Listeria monocytogenes. In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Methanobrevibacter spp. In one embodiment, the Methanobrevibacter spp. is Methanobrevibacter smithii. Other genes encoding bile salt hydrolase enzymes are well-known to one of ordinary skill in the art and described in, for example, Jones et al., PNAS, 105(36):13580-13585 (2008) and WO2014/198857. Table 11A lists non-limiting examples of bile salt hydrolases.

TABLE 11A
Bile Salt Hydrolases
Gene or Operon      Sequence
Bile salt hydrolase ATGTGTACTGCCATAACTTATCAATCTTATAATAATTACTTC
from Lactobacillus  GGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTT
plantarum           ACGATTACGCCTAGAAAATATCCACTAGTATTTCGTAAGGTG
SEQ ID NO: 90       GAGAACTTAGATCACCATTATGCAATAATTGGAATTACTGCT
                    GATGTAGAAAGCTATCCACTTTACTACGATGCGATGAATGAA
                    AAAGGCTTGTGTATTGCGGGATTAAATTTTGCAGGTTATGCT
                    GATTATAAAAAATATGATGCTGATAAAGTTAATATCACACCA
                    TTTGAATTAATTCCTTGGTTATTGGGACAATTTTCAAGTGTT
                    AGAGAAGTGAAAAAGAACATACAAAAACTAAACTTGGTTAAT
                    ATTAATTITAGTGAACAATTACCATTATCACCGCTACATTGG
                    TTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGTGIC
                    AAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTA
                    ACAAACAATCCTAATTTTGACTACCAATTATTTAATTTGAAC
                    AACTATCGTGCCTTATCAAATAGCACACCCCAAAATAGTITT
                    TCGGAAAAAGTGGATTTAGATAGTTATAGTAGAGGAATGGGC
                    GGACTAGGATTACCTGGAGACTTGTCCTCAATGICTAGATTT
                    GTCAGAGCCGCTTTTACTAAATTAAACTCGTTGTCGATGCAG
                    ACAGAGAGTGGCAGTGTTAGTCAGTTTTTCCATATACTAGGG
                    TCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGA
                    AAGTACGAATATACAATCTATTCTTCTTGTTGTGATATGGAC
                    AAAGGAGTTTATTACTATAGAACTTATGACAATAGTCAAATT
                    AACAGTGTCAGTTTAAACCATGAGCACTTGGATACGACTGAA
                    TTAATTTCTTATCCATTACGATCAGAAGCACAATACTATGCA
                    GTTAACTAA
Bile salt hydrolase MCTAITYQSYNNYFGRNFDYEISYNEMVTITPRKYPLVFRKV
protein from        ENLDHHYAIIGITADVESYPLYYDAMNEKGLCIAGLNFAGYA
Lactobacillus       DYKKYDADKVNITPFELIPWLLGQFSSVREVKKNIQKLNLVN
plantarum           INFSEQLPLSPLHWLVADKQESIVIESVKEGLKIYDNPVGVL
SEQ ID NO: 91       TNNPNFDYQLFNLNNYRALSNSTPQNSFSEKVDLDSYSRGMG
                    GLGLPGDLSSMSRFVRAAFTKLNSLSMQTESGSVSQFFHILG
                    SVEQQKGLCEVTDGKYEYTIYSSCCDMDKGVYYYRTYDNSQI
                    NSVSLNHEHLDTTELISYPLRSEAQYYAVN
Bile salt hydrolase ATGTGTACTGCTGCAAATTATTTAACAAAATGCCATTATTTT
from                GGCCGTAATTTTGACTATGAAATTTCATATAATGAAAGAGTA
Methanobrevibacter  ACGATAACTCCTAGAAACTATCCTTTAATATTCAGGGATACT
smithii 3142        GAGGACATTGAAAATCATTATGGGATTATTGGCATAGCTGCA
SEQ ID NO: 92       GGTATTGATGAATATCCTTTGTATTATGATGCATGTAATGAG
                    AAAGGATTAGCTATGGGGGGATTAAACTTTCCGGATTACTGT
                    GACTACAAACCACTAGATAAATCTAAAGTTAACATAGCTTCT
                    TITGAGATTATTCCATATATATTATCTCAAGCAAAAACCATC
                    AGTGATGCCGAAAGGTTATTGGAAAACTTAAATATTTCAGAT
                    GAGAAATTTTCCGCCCAGTTGCCTCCATCTCCACTTCATTGG
                    ATTATTTCAGATAGGAATGCTTCAATTGTTGTAGAGGTTGTA
                    GAGGAAGGACTGGATATTTATGATAATCCTGTAGGAGTTITA
                    ACAAACAACCCTCCTTTTGATAAACAGCTATTTAATTTAAAT
                    AATTATATGGCATTATCAAACAGAACGCCTGAAAATACCTTT
                    GGAGGCAATTTGGATTTGGCAACTTATAGTCGGGGAATGGGT
                    TCAATTGGTCTTCCGGGGGATGTTTCTTCACAGTCCCGTTTT
                    GTAAAAGCAGCTTTTGTTAAAGAAAATTCCGTTTCCGGAGAT
                    TCTGAAAAAGAAAGTGTGTCTCAGTTTTTCCATATTCTGGCA
                    TCTGTTGAACAGCAAAAAGGATGTACGTTAGTGGAAGAACCT
                    GATAAATTTGAGTATACTATTTATTCAGACTGTTACAATACA
                    GATAAGGGAATATTGTATTATAAAACATATGATGGTCCTCAA
                    ACATCTGTTAATATACATGATGAGGATTTGGAAACCAATCAG
                    TTAATTAATTTTGAGTTGGTTGATTAA
Bile salt hydrolase MCTAANYLTKCHYFGRNFDYEISYNERVTITPRNYPLIFRDT
protein from        EDIENHYGIIGIAAGIDEYPLYYDACNEKGLAMGGLNFPDYC
Methanobrevibacter  DYKPLDKSKVNIASFEIIPYILSQAKTISDAERLLENLNISD
smithii 3142        EKFSAQLPPSPLHWIISDRNASIVVEVVEEGLDIYDNPVGVL
SEQ ID NO: 93       TNNPPFDKQLFNLNNYMALSNRTPENTFGGNLDLATYSRGMG
                    SIGLPGDVSSQSRFVKAAFVKENSVSGDSEKESVSQFFHILA
                    SVEQQKGCTLVEEPDKFEYTIYSDCYNTDKGILYYKTYDGPQ
                    TSVNIHDEDLETNQLINFELVD
Bile salt hydrolase ATGGTTATGAAAAAGATTTTGATAGCTTTGGCCTTATTGCTG
from Bacteroides    ACAGGCATTGCAAGCGGATCGGCATGTACCGGTATTTCATTC
vulgatus            CTCGCTGAAGATGGCGGATATGTGCAGGCACGTACTATAGAG
SEQ ID NO: 94       TGGGGGAACAGTTATCTTCCGAGTGAATATGTTATTGTTCCC
                    AGAGGACAGGATTTGGTATCTTATACTCCAACGGGTGTAAAT
                    GGCTTGAGATTTCGGGCTAAATATGGTCTGGTAGGACTGGCT
                    ATCATTCAGAAAGAGTTTGTGGCTGAAGGACTGAATGAAGTA
                    GGGCTTTCGGCTGGATTGTTTTATTTTCCCCATTATGGGAAG
                    TATGAAGAATATGATGAGGCTCAAAATGCAATTACTTTGTCG
                    GATTTGCAGGTGGTGAACTGGATGCTTTCCCAATTTGCTACT
                    ATAGACGAAGTGAGAGAAGCTATAGAAGGGGTGAAGGTGGTG
                    TCTCTTGATAAACCTGGTAAAAGTTCTACGGTACATTGGCGC
                    ATTGGCGATGCTAAAGGAAATCAAATGGTGTTGGAATTTGTA
                    GGTGGTGTTCCTTATTTTTATGAAAATAAAGTAGGAGTACTC
                    ACCAATTCTCCCGATTTTCCATGGCAGGTGATTAACTTGAAT
                    AATTATGTAAATCTATATCCGGGAGCTGTCACTCCACAGCAA
                    TGGGGTGGGGTGACTATTTTCCCTTTTGGCGCAGGTGCCGGA
                    TTTCATGGTATTCCGGGGGATGTAACTCCTCCATCCCGTTTT
                    GTTCGTGTAGCGTTTTATAAGGCAACAGCTCCGGTGTGTCCT
                    ACAGCGTATGACGCTATATTACAAAGCTTTCATATCCTGAAT
                    AATTTTGATATTCCTATTGGTATAGAATATGCGTTAGGGAAA
                    GCACCTGATATTCCTAGTGCCACACAATGGACTTCGGCTATT
                    GAITTGACAAACAGGAAAGTGTATTATAAAACAGCATACAAT
                    AACAATATTCGTTGTATTAGTATGAAGAAGATTGATTTTGAT
                    AAAGTGAAGTATCAGTCGTATCCATTGGATAAGGAGTTGAAA
                    CAGCCTGTAGAAGAGATTATTGTGAAATAG
Bile salt hydrolase MVMKKILIALALLLTGIASGSACTGISFLAEDGGYVQARTIE
protein from        WGNSYLPSEYVIVPRGQDLVSYTPTGVNGLRFRAKYGLVGLA
Bacteroides         IIQKEFVAEGLNEVGLSAGLFYFPHYGKYEEYDEAQNAITLS
vulgatus            DLQVVNWMLSQFATIDEVREAIEGVKVVSLDKPGKSSTVHWR
SEQ ID NO: 95       IGDAKGNQMVLEFVGGVPYFYENKVGVLTNSPDFPWQVINLN
                    NYVNLYPGAVTPQQWGGVTIFPFGAGAGFHGIPGDVTPPSRF
                    VRVAFYKATAPVCPTAYDAILQSFHILNNFDIPIGIEYALGK
                    APDIPSATQWTSAIDLTNRKVYYKTAYNNNIRCISMKKIDFD
                    KVKYQSYPLDKELKQPVEEIIVK
Bile salt hydrolase ATGTGCACTGGTGTCCGTTTCTCCGATGATGAGGGCAACAC
from                CTATTTCGGCCGTAATCTCGACTGGAGTTTCTCATATGGGG
Bifidobacterium     AGACCATCCTGGTTACTCCGCGCGGCTACCACTATGACACG
longum              GTGTTTGGTGCGGGCGGCAAGGCGAAGCCGAACGCGGTGAT
SEQ ID NO: 96       CGGCGTGGGTGTGGTCATGGCCGATAGGCCGATGTATTTCG
                    ACTGCGCCAATGAACATGGTCTGGCCATCGCCGGCTTGAAT
                    TTCCCCGGCTACGCCTCGTTCGTCCACGAACCGGTCGAAGG
                    CACGGAAAACGTCGCCACGTTCGAATTTCCGCTGTGGGTGG
                    CGCGTAATTTCGACTCCGTCGACGAGGTCGAGGAGGCGCTC
                    AGGAACGTGACGCTCGTCTCCCAGATCGTGCCGGGACAGCA
                    GGAGTCTCTGCTGCACTGGTTCATCGGCGACGGCAAGCGCA
                    GCATCGTCGTCGAGCAGATGGCCGATGGCATGCACGTGCAT
                    CATGATGACGTCGATGTGCTGACCAATCAGCCGACGTTCGA
                    CTTCCATATGGAAAACCTGCGCAACTACATGTGCGTCAGCA
                    ACGAGATGGCCGAACCGACTTCATGGGGCAAGGCCTCCTTG
                    ACCGCCTGGGGTGCGGGTGTGGGCATGCATGGCATCCCGGG
                    CGACGTGAGTTCCCCGTCGCGCTTCGTTCGTGTGGCCTACA
                    CCAACGCGCATTACCCGCAGCAGAACGATGAAGCCGCCAAT
                    GTGTCGCGCCTGTTCCACACCCTCGGCTCCGTGCAGATGGT
                    GGACGGCATGGCGAAGATGGGCGACGGCCAGTTCGAACGCA
                    CGCTGTTCACCAGCGGATATTCGTCCAAGACCAACACCTAT
                    TACATGAACACCTATGATGACCCCGCCATCCGTTCCTACGC
                    CATGGCCGATTACGATATGGATTCCTCGGAGCTCATCAGCG
                    TCGCCCGATGA
Bile salt hydrolase MCTGVRFSDDEGNTYFGRNLDWSFSYGETILVTPRGYHYDTV
protein from        FGAGGKAKPNAVIGVGVVMADRPMYFDCANEHGLAIAGLNFP
Bifidobacterium     GYASFVHEPVEGTENVATFEFPLWVARNFDSVDEVEEALRNV
longum              TLVSQIVPGQQESLLHWFIGDGKRSIVVEQMADGMHVHHDDV
SEQ ID NO: 97       DVLTNQPTFDFHMENLRNYMCVSNEMAEPTSWGKASLTAWGA
                    GVGMHGIPGDVSSPSRFVRVAYTNAHYPQQNDEAANVSRLFH
                    TLGSVQMVDGMAKMGDGQFERTLFTSGYSSKTNTYYMNTYDD
                    PAIRSYAMADYDMDSSELISVAR
Bile salt hydrolase ATGTGTACGTCAATAACTTATACAACGAAGGATCACTATTT
from Listeria       TGGAAGGAATTTCGATTATGAACTTTCTTACAAAGAAGTTG
monocytogenes       TGGTTGTTACGCCGAAAAATTACCCGTTCCATTTTCGCAAG
SEQ ID NO: 98       GTAGAGGATATAGAGAAGCATTATGCACTTATTGGTATTGC
                    TGCTGTGATGGAAAACTACCCGTTGTATTACGATGCTACCA
                    ATGAAAAAGGCCTTAGTATGGCAGGACTCAATTTCTCAGGA
                    AATGCGGATTACAAGGATTTTGCAGAAGGTAAGGACAATGT
                    GACCCCCTTTGAATTTATTCCGTGGATTCTTGGTCAATGCG
                    CTACTGTAAAAGAAGCAAGAAGATTACTTCAGAGAATCAAT
                    CTCGTGAATATTAGTTTTAGTGAAAATTTACCGCTGTCTCC
                    ATTACATTGGTTGATGGCTGATCAAACAGAATCTATTGTAG
                    TGGAATGTGTGAAAGATGGACTTCACATTTATGATAATCCT
                    GTTGGCGTGTTAACAAATAATCCAACATTTGATTACCAACT
                    ATTTAATTTAAACAATTATCGCGTTCTTTCGAGTGAAACCC
                    CAGAAAATAATTTTTCCAAAGAGATTGATTTGGATGCTTAT
                    AGTCGTGGGATGGGCGGAATTGGCTTACCTGGTGATTTATC
                    TTCTATGTCTCGTTTTGTGAAAGCAACTTTTACCAAATTGA
                    ATTCTGTTTCAGGTGATTCTGAATCAGAAAGTATTAGCCAA
                    TTTTTCCATATTTTAGGCTCGGTGGAACAACAAAAAGGTCT
                    TTGTGATGTTGGTGGGGGAAAATACGAGCATACTATTTATT
                    CCTCGTGTTGCAATATCGATAAAGGAATTTATTATTATAGA
                    ACATACGGAAACAGTCAAATTACTGGTGTGGATATGCACCA
                    AGAGGATTTAGAGAGCAAAGAACTAGCTATTTATCCACTCG
                    TCAATGAGCAACGACTAAACATTGTTAACAAATAA
Bile salt hydrolase MCTSITYTTKDHYFGRNFDYELSYKEVVVVTPKNYPFHFRKV
protein from        EDIEKHYALIGIAAVMENYPLYYDATNEKGLSMAGLNFSGNA
Listeria            DYKDFAEGKDNVTPFEFIPWILGQCATVKEARRLLQRINLVN
monocytogenes       ISFSENLPLSPLHWLMADQTESIVVECVKDGLHIYDNPVGVL
SEQ ID NO: 99       TNNPTFDYQLFNLNNYRVLSSETPENNFSKEIDLDAYSRGMG
                    GIGLPGDLSSMSRFVKATFTKLNSVSGDSESESISQFFHILG
                    SVEQQKGLCDVGGGKYEHTIYSSCCNIDKGIYYYRTYGNSQI
                    TGVDMHQEDLESKELAIYPLVNEQRLNIVNK
Bile salt hydrolase ATGTGTACAGGATTAGCCTTAGAAACAAAAGATGGATTACAT
from Clostridium    TTGTTTGGAAGAAATATGGATATTGAATATTCATTTAATCAA
perfringens         TCTATTATATTTATTCCTAGGAATTTTAAATGTGTAAACAAA
SEQ ID NO: 100      TCAAACAAAAAAGAATTAACAACAAAATATGCTGTTCTTGGA
                    ATGGGAACTATTTTTGATGATTATCCTACCTTTGCAGATGGT
                    ATGAATGAAAAGGGATTAGGGTGTGCTGGCTTAAATTTCCCT
                    GTTTATGTTAGCTATTCTAAAGAAGATATAGAAGGTAAAACT
                    AATATTCCAGTATATAATTTCTTATTATGGGTTTTAGCTAAT
                    TTTAGCTCAGTAGAAGAGGTAAAGGAAGCATTAAAAAATGCT
                    AATATAGTGGATATACCTATTAGCGAAAATATTCCTAATACA
                    ACTCTTCATTGGATGATAAGCGATATAACAGGAAAGTCTATT
                    GTGGTTGAACAAACAAAGGAAAAATTAAATGTATTTGATAAT
                    AATATTGGAGTATTAACTAATTCACCTACTTTTGATTGGCAT
                    GTAGCAAATTTAAATCAATATGTAGGTTTGAGATATAATCAA
                    GTTCCAGAATTTAAGTTAGGAGATCAATCTTTAACTGCTTTA
                    GGTCAAGGAACTGGTTTAGTAGGATTACCAGGGGACTTTACA
                    CCTGCATCTAGATTTATAAGAGTAGCATTTTTAAGAGATGCA
                    ATGATAAAAAATGATAAAGATTCAATAGACTTAATTGAATTT
                    TTCCATATATTAAATAATGTTGCTATGGTAAGAGGATCAACT
                    AGAACTGTAGAAGAAAAAAGTGATCTTACTCAATATACAAGT
                    TGCATGTGTTTAGAAAAAGGAATTTATTATTATAATACCTAT
                    GAAAATAATCAAATTAATGCAATAGACATGAATAAAGAAAAC
                    TTAGATGGAAATGAAATTAAAACATATAAATACAACAAAACT
                    TTAAGTATTAATCATGTAAATTAG
Bile salt hydrolase MCTGLALETKDGLHLFGRNMDIEYSFNQSIIFIPRNFKCVNK
protein from        SNKKELTTKYAVLGMGTIFDDYPTFADGMNEKGLGCAGLNFP
Clostridium         VYVSYSKEDIEGKTNIPVYNFLLWVLANFSSVEEVKEALKNA
perfringens         NIVDIPISENIPNTILHWMISDITGKSIVVEQTKEKLNVFDN
SEQ ID NO: 101      NIGVLTNSPTFDWHVANLNQYVGLRYNQVPEFKLGDQSLTAL
                    GQGTGLVGLPGDFTPASRYIRVAFLRDAMIKNDKDSIDLIEF
                    FHILNNVAMVRGSTRTVEEKSDLTQYTSCMCLEKGIYYYNTY
                    ENNQINAIDMNKENLDGNEIKTYKYNKTLSINHVN
Bile salt hydrolase ATGTGTACGTCTATTACTTATGTAACAAGTGATCATTATTTT
from Enterococcus   GGAAGGAATTTTGATTATGAAATATCTTACAATGAAGTAGTT
faecium             ACTGTTACTCCAAGAAATTATAAGTTGAATTTTCGAAAGGTA
SEQ ID NO: 102      AATGATTTGGATACTCATTATGCAATGATTGGTATTGCCGCT
                    GGTATAGCTGACTACCCTCTTTATTACGATGCGACAAATGAA
                    AAAGGATTGAGTATGGCTGGGCTAAATTTTTCTGGGTATGCT
                    GATTATAAAGAAATACAAGAAGGGAAAGACAATGTATCTCCT
                    TTTGAATTTATTCCTTGGATTTTAGGACAATGCTCAACAGTA
                    GGAGAAGCTAAAAAATTGTTAAAAAATATCAATTTAGCAAAT
                    ATAAATTATAGTGACGAACTTCCTTTATCCCCTTTACATTGG
                    CTATTAGCTGATAAAGAAAAATCAATTGTCATTGAAAGTATG
                    AAAGATGGACTTCATATATATGATAACCCTGTGGGCGTTCTT
                    ACCAATAATCCTTCATTTGACTATCAATTATTTAATTTAAAC
                    AATTATCGTGTCTTATCGAGTGAAACTCCTAAAAATAATTTT
                    TCAAATCAAATAAGTTTGAATGCCTATAGCCGCGGTATGGGA
                    GGGATAGGCTTGCCTGGAGATTTATCCTCAGTATCTCGTTTT
                    GTTAAAGCGACTTTTACGAAGCTGAATTCTGTATCTGGAGAT
                    TCAGAGTCAGAAAGTATTAGTCAATTTTTCCATATCTTAGGT
                    TCAGTAGAACAACAAAAAGGTTTGTGTGATGTAGGTGATGGA
                    AAATATGAATATACAATTTATTCTTCTTGTTGCAATGTTGAC
                    AAAGGAATCTATTATTATCGAACATATGAAGACAGTCAAATT
                    ACTGCAATTGATATGAATAAAGAAGACTTAGATAGTCATAAG
                    TTAATTAGTTATCCAATTATAGAAAAACAACAAATTAAATAT
                    ATAAATTAG
Bile salt hydrolase MCTSITYVTSDHYFGRNFDYEISYNEVVTVTPRNYKLNFRKV
protein from        NDLDTHYAMIGIAAGIADYPLYYDATNEKGLSMAGLNFSGYA
Enterococcus        DYKEIQEGKDNVSPFEFIPWILGQCSTVGEAKKLLKMINLAN
faecium             INYSDELPLSPLHWLLADKEKSIVIE8MKDGLHIYDNPVGVL
SEQ ID NO: 103      TNWPSFDYQLFNLNNYRVLSSETPKNNFSNQISLNAYSRGMG
                    GIGLPGDLSSVSRFVKATFTKLNSVSGDSESESISQFFHILG
                    SVEQQKGLCDVGDGKYEYTIYSSCCNVDKGIYYYRTYEDSQI
                    TAIDMNKEDLDSHKLISYPIIEKQQIKYIN
Bile salt hydrolase AAGAGAAAAATATGTGTACATCAATTATATTCAGTCCCAAAG
A from              ATCATTACTTTGGTCGTAACCTTGATTTAGAAATTACTTTTG
Lacotbacillus       GTCAACAAGTTGTTATTACGCCACGCAATTACACTTTTAAAT
acidophilus         TCCGTAAGATGCCCAGTTTAAAAAAGCACTATGCAATGATTG
SEQ ID NO: 104      GTATCTCATTAGATATGGATGATTATCCCCTATATTTCGACG
                    CTACAAATGAAAAAGGTTTAGGTATGGCCGGACTCAACTATC
                    CAGGAAATGCTACATATTATGAAGAAAAAGAAAATAAAGATA
                    ATATTGCTTCCTTTGAATTCATCCCTTGGATTTTAGGACAGT
                    GTAGCACTATTAGCGAAGTAAAGGATTTACTTAGCAGAATCA
                    ACATCGCCGATTTAAATTTCAGCGAAAAAATGCAAGCCTCCT
                    CTCTTCACTGGCTTATTGCAGATAAAACAGGTACATCATTAG
                    TTGTTGAAACAGACAAAGATGGAATGCATATTTATGATAATC
                    CAGTTGGCTGCTTAACTAATAATCCACAATTTCCAAAGCAAT
                    TATTCAATTTAAATAACTATGCTGACGTATCTCCAAAAATGC
                    CTAAAAATAACTTCTCAGATAAAGTAAATATGGCTGGCTACA
                    GCCGTGGATTAGGGTCTCACAACTTACCAGGTGGAATGGATT
                    CTGAATCACGTTTTGTCAGAGTAGCTTTCAATAAATTTAATG
                    CTCCAATTGCTGAAACCGAAGAAGAAAATATTGATACTTACT
                    TCCACATTTTACATTCGGTTGAACAACAAAAGGGACTGGATG
                    AAGTTGGTCCAAACTCATTTGAATATACAATTTATTCTGATG
                    GAACTAACTTAGACAAAGGTATTTTCTACTACACCACTTATT
                    CAAACAAACAAATTAACGTTGTTGATATGAATAAAGAAGATC
                    TAGATAGCAGCAATTTGATCACTTATGATATGCTTGATAAAA
                    CTAAATTTAACCATCAAAACTAA
Bile salt hydrolase MCTSIIFSPKDHYFGRNLDLEITFGQQVVITPRNYTFKFRKM
A protein from      PSLKKHYAMIGISLDMDDYPLYYDATNEKGLGMAGINYPGNA
Lacotbacillus       TYYEEKENKDNIASFEFIPWILGQCSTISEVKDLLSRINIAD
acidophilus         LNFSEKMQASSLHWLIADKIGTSLVVETDKDGMHIYDNPVGC
SEQ ID NO: 105      LTNNPQFPKQLFNLNNYADVSPKMPKNNFSDKVNMAGYSRGL
                    GSHNLPGGMDSESRFVRVAFNKFNAPIAETEEENIDTYFHIL
                    HSVFOQKGLDEVGPNSFEYTIYSDGINLDKGIFYYTTYSNKQ
                    INVVDMNKFDLDSSNIITYDPILDKIKFNHQN
Bilesalthydrolase   AGAAAGCGTGCAGTAAATGTGTACATCAATTTGTTATAATC
B from              CTAACGATCATTATTTTGGTAGAAATCTTGACTATGAAATT
Lacotbacillus       GCTTATGGTCAAAAAGTAGTCATTGTACCAAGAAACTACGA
acidophilus         ATTTAAGTATAGAGAAATGCCCTCTCAAAAGATGCATTATG
SEQ ID NO: 106      CTTTTATCGGAGTATCTGTAGTTAATGATGATTATCCATTA
                    TTATGTGATGCAATTAATGAAAAGGGGCTTGGTATTGCAGG
                    ATTAAATTTTCAAGGTCCTAATCATTACTTTCCTAAAATCG
                    AAGGTAAGAAGAATATTGCTTCTTTTGAATTAATGCCATAC
                    TTATTAAGTAATTGTGAAAATACTGACGATGTTAAAGAAAT
                    CTTAGATAATGCAAATATTTTAAATATTAGCTTTTCAGCAA
                    ATTATCCTGCAGCTGATTTACATTGGATTTTAAGTGATAAA
                    GCTGGTAAGAGTATCGTAGTTGAATCAACCAATTCAGGTTT
                    ACATATTTATGATAATCCAGTGAATGTCTTAACTAACAATC
                    CTGAATTTCCGGATCAATTAATTAAATTAAGTGACTACGCC
                    GACGTTACTCCACATAATCCTAAGAATACATTGGTTCCTAA
                    TGTTGATCTTAATCTATATAGTAGAGGCTTAGGTACTCACC
                    ACTTACCTGGTGGAATGGATTCTAGCTCTCGATTTGTTAAG
                    GTAGCTTTTGTCTTGGCACACACTCCACAAGGAAAAAATGA
                    AGTGGAAAATGTTACTAATTATTTCCATATTCTGCATTCAG
                    TAGAACAACCTGATGGTTTAGATGAAGTAGAAGATAATCGC
                    TATGAATATACTATGTATACAGATTGTATGAACTTAGATAA
                    AGGTATTTTGTACTTTACTACTTATGACAATAATCGGATTA
                    ATGCAGTAGATATGCATAAAGCAGATTTAGATTCAGAAGAT
                    TTAATCTGCTACGATTTGTTTAAGAAACAAGATATTGAATA
                    TATGAATTAA
Bile salt hydrolase MCTSICYNPNDHYFGRNLDYEIAYGQKVVIVPRNYEFKYREM
B protein from      PSQKMHYAFIGVSVVNDDYPLLCDAINEKGLGIAGLNFQGPN
Lacotbacillus       HYFPKIEGKKNIASFELMPYLLSNCENTDDVKEILDNANILM
acidophilus         ISFSANYPAADLHWILSDKAGKSIVVESTNSGLHIYDNPVNV
SEQ ID NO: 107      LTNNPEFPDQLIKLSDYADVTPHNPKNTLVPNVDLNLYSRGL
                    GTHHLPGGMDSSSRFVKVAFVLAHTPQGKNEVENVTNYFHIL
                    HSVEQPDGLDEVEDNRYEYTMYTDCMNLDKGILYFTTYDNNR
                    INAVDMHKADLDSEDLICYDLFKKQDIEYMN
Bile salt hydrolase ATGGAAACGAAAAGCTCTCTCTGGAAATCATCGCGCCGCGT
from Brucella       GCTTGCACATGGGGCTGCAACTGTTCTGGTCGCGGCGGGCC
abortus             TTATCGTTCCCCAGGCGGCTATGGCTTGCACGAGCTTCGTT
SEQ ID NO: 108      CTGCCGACGAGCGACGGTGGTATGGTCTATGGTCGCACGAT
                    GGAATTCGGGTTCAATCTCAAATCCGACATGATTGCCATTC
                    CGCGCAATTACACCATCACGGCAAGCGGGCCGGACGGTGCT
                    GCGGGCAAGAAATGGAAGGGCAAATATGCCACGATCGGCAT
                    GAATGCTTTTGGTATCGTCGCTCTCACCGACGGTATGAACG
                    AGAAGGGGCTTGCAGGCGGGCTTCTCTATTTCCCGGAATAT
                    GCCAAGTATCAGGACCCATCCACGGCGAAGCCGGAAGACAG
                    CCTCGCTCCGTGGGATTTCCTGACCTGGGCGCTGGCCAATT
                    TTTCGACAGTGGCCGAAGTCAAGGATGCTTTGAGCACCATT
                    TCCATCGTCGATGTGAAACAAAAGGACCTGGGATTTACCCC
                    GCCCGCTCACTACACGCTGCATGATGCGACCGGCGCATCCA
                    TCGTGATCGAACCGATCGACGGCAAGCTCAAGGTTTACGAC
                    AACAAGCTCGGTGTCATGACCAATTCGCCGTCTTTCGACTG
                    GCACATGACCAATCTGCGCAACTATGTCTATCTCTCGCGTG
                    AAAATCCGAAGCCGTTGCAGATCCTTGGCGAGACGATCCAG
                    TCATTCGGGCAAGGCGCCGGTATGCATGGTATTCCGGGCGA
                    CACCACGCCGCCATCGCGTTTCGTGCGTGCAAGCGCCTACG
                    TCCTTTCCGCCAAGAAGGTGCCGAGCGGCCTTGAAAGCGTG
                    CGGCTGGCCGAGCATATTGCCAATAACTTCGACATTCCAAA
                    GGGATGGAGCGAAGAGCAGAATATGTTTGAATATACCCAGT
                    GGACCGCCTTTGCGGACATGAAGAACGATGTCTATTACATC
                    AAGACCTATGACGATCAGGTTCTGCGCAGCTTCAGCTTCAA
                    GGATTTTGATGTCGATAGCAAAGATATTCTAACGATCAAGT
                    TCGAGCCAAAACTGGACGCGCCGTCACTGAAAAAGTAA
Bile salt hydrolase METKSSLWKSSRRVLAHGAATVLVAAGLIVPQAAMACTSFVL
protein from        PTSDGGMVYGRTMEFGFNLKSDMIAIPRNYTITASGPDGAAG
Brucella abortus    KKWKGKYATIGMNAFGIVALTDGMNEKGLAGGLLYFPEYAKY
SEQ ID NO: 109      QDPSTAKPEDSLAPWDFLTWALANFSTVAEVKDALSTISIVD
                    VKQKDLGFTPPAHYTLHDATGASIVIEPIDGKLKVYDNKLGV
                    MTNSPSFDWHMTNLRNYVYLSRENPKPLQILGETIQSFGQGA
                    GMHGIPGDTTPPSRFVRASAYVLSAKKVPSGLESVRLAEHIA
                    NNFDIPKGWSEEQNMFEYTQWTAFADMKNDVYYIKTYDDQVL
                    RSFSFKDFDVDSKDILTIKFEPKLDAPSLKK

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 90. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 90. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 90.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 92. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 92. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 92.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 94 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 94. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 93. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 94. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 94. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 94.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 96 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 96. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 96. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 96.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 98. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 98. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 98.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 100. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 100.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 102. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 102. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 102.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 104. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 104. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 104.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 106. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 106. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 106.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 108. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 108. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 108.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109.

In one embodiment, the gene encoding the bile salt hydrolase enzyme is directly operably linked to a first promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is indirectly operably linked to a first promoter. In one embodiment, the gene encoding bile salt hydrolase enzyme is operably linked to a promoter that it is not nauturally linked to in nature.

In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the bile salt hydrolase enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose .

The gene encoding the bile salt hydrolase enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the bile salt hydrolase is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding a bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example, E. coli comprises a native bile salt hydrolase gene.

In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a low-copy plasmid. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile salt hydrolase enzyme, thereby increasing the catabolism of bile salts.

Transporters of Bile Salts and Bile Acids

The uptake of bile salts into the Lactobacillus and Bifidobacterium has been found to occur via the bile salt transporters CbsT1 and CbsT2 (see, e.g., Elkins et al., Microbiology, 147(Pt. 12):3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference). The uptake of bile acids into the Neisseria meningitides has been found to occur via the bile acid sodium symporter ASBT (see, e.g., Hu et al., Nature, 478(7369):408-411 (2011), the contents of which are expressly incorporated herein by reference. Other proteins that mediate the import of bile salts or acids into cells are well known to those of skill in the art. For the purposes of this invention, a bile salt transporter includes bile salt importers and bile acid symporters.

Bile salt transporters, e.g., bile salt importers or bile acid symporters, may be expressed or modified in the bacteria in order to enhance bile salt or acid transport into the cell. Specifically, when the transporter of bile salts is expressed in the recombinant bacterial cells, the bacterial cells import more bile salts into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a transporter of bile salts may be used to import bile salts into the bacteria so that any gene encoding a bile salt hydrolase (BSH) enzyme expressed in the organism can be used to treat disorders associated with bile salts, such as cardiac disease, metabolic disease, liver disease, cancer, and C. difficile infection. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt and a heterologous gene encoding a bile salt hydrolase (BSH) enzyme.

Thus, in some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a transporter of a bile salt operably linked to the first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding transporter of a bile salt operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters. In some embodiments the gene encoding at least one bile salt hydrolase enzyme and/or the heterologous gene encoding transporter of a bile salt are operably linked to a promoter that it is not naturally linked to in nature.

In one embodiment, the bacterial cell comprises a gene encoding a transporter of a bile salt from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding transporter of a bile salt. In some embodiments, the at least one native gene encoding atransporter of a bile salt is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding a transporter of a bile salt. In yet another embodiment, the bacterial cell comprises a copy of a gene encoding a native transporter of a bile salt, as well as at least one copy of a heterologous gene encoding a transporter of a bile salt from a different bacterial species. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of the heterologous gene encoding a tarnsporter of a bile salt. In one embodiment, the bacterial cell comprises multiple copies of the heterologous gene encoding a transporter of a bile salt.

In some embodiments, the transporterof a bile salt is encoded by a transporter of a bile salt gene derived from a bacterial genus or species, including but not limited to, Lactobacillus. In some embodiments, the transporterof a bile salt gene is derived from a bacteria of the species Lactobacillus johnsonni strain 100-100.

The present disclosure further comprises genes encoding functional fragments of a transporter of a bile salt or functional variants of a transporter of a bile salt. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a transporter of a bile salt relates to an element having qualitative biological activity in common with the wild-type transporter of a bile salt from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated transporter of bile salt protein is one which retains essentially the same ability to import the bile salt into the bacterial cell as does the transporter protein from which the functional fragment or functional variant was derived. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional fragment of a transporter of a bile salt. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional variant of a transporter of a bile salt.

Assays for testing the activity of a transporter of a bile salt, a functional variant of a transporter of a bile salt, or a functional fragment of a transporter of a bile salt are well known to one of ordinary skill in the art. For example, bile salt import can be assessed as described in Elkins et al., Microbiology, 147:3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference.

In one embodiment, the gene(s) encoding the transporter of a bile salt have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the transporter of a bile salt have been codon-optimized for use in Escherichia coli.

The present disclosure also encompasses genes encoding a transporter of a bile salt comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.

In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting increased bile salt transport are selected; and the mutagenized a gene encoding a transporter of a bile salt is isolated and inserted into the bacterial cell. In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting decreased bile salt transport are selected; and the mutagenized a gene encoding a transporter of the bile salt is isolated and inserted into the bacterial cell. The transporter modifications described herein may be present on a plasmid or chromosome. Non-limiting examples of bile salt transporters, which are encoded in the genetically engineered bacteria, are in Table 11B.

TABLE 11B
Bile Salt Transport and Export Sequences
Description   Sequence
cbsT1 from    ATGTCGACCACACCGACACAGCCATCATCACGAAAACAG
Lactobacillus GCTGTTTACCCGTACTTGATCGTGCTGTCGGGCATCGTCT
johnsonii     TCACGGCCATCCCGGTATCGCTGGTCTGCAGTTGCGCAGG
SEQ ID NO:    TATCTTCTTCACGCCTGTCAGCAGCTACTTCCATGTTCCCA
110           AGGCCGCATTCACCGGATATTTCAGCATATTCAGCATCAC
              CATGGTCGCCTTCCTGCCGGTGGCCGGATGGCTGATGCAC
              CGCTACGATCTGCGCATCGTACTGACCGCAAGCACCGTCC
              TGGCTGGACTGGGCTGCCTGGGTATGTCCCGATCATCCGC
              CATGTGGCAGTTCTATCTATGCGGAGTGGTTCTGGGAATC
              GGCATGCCGGCCGTCCTCTATCTGTCAGTGCCAACACTCA
              TCAACGCCTGGTTCCGCAAGCGGGTCGGGTTCTTCATCGG
              CCTGTGCATGGCCTTCACCGGCATAGGCGGCGTGATCTTC
              AACCAGATAGGCACCATGATCATCAGATCCGCCCCTGAT
              GGATGGAGGCGGGGATATCTGGTTTTCGCTATTCTCATCC
              TGGTGATCACCCTGCCCTTCACCATTTTCGTCATTCGCAG
              CACACCCGAACAGATGGGTCTGCATCCCTACGGCGCCGA
              CCAGGAGCCTGATGCAGCTGAGACGGCCACCAATAGTGC
              AGGCACCGGGAGCAAAGACCAAAAGAGTCCTGAGCCTGC
              AGCGTCAACCGTAGGCATGACTGCCTCCCAGGCCTTGCGC
              TCCCCTGCCTTCTGGGCGCTGGCGCTCTTCTGCGGTCTGA
              TCACCATGAATCAGACCATTTACCAGTTCCTGCCCTCCTA
              CGCGGCATCCCTGCCATCCATGGCAGCCTACACGGGACT
              GATCGCCTCCTCCTGCATGGCCGGCCAGGCCATCGGCAA
              GATCATCCTGGGCATGGTCAACGACGGCAGCATCGTAGG
              CGGTCTCTGTCTGGGCATCGGCGGCGGCATTCTCGGCGTC
              TGCCTCATGGTCGCCTTCCCCGGATTGCCCGTGCTCCTCCT
              GCTGGGAGCCTTTGCCTTCGGCCTTGTCTACGCCTGCACT
              ACTGTGCAGACACCAATCCTGGTTACAGCGGTCTTCGGCT
              CGCGCGACTACACCAACATCTATGCACGTATCCAGATGGT
              TGGGTCCCTAGCCTCGGCCTTCGCAGCTCTCTTCTGGGGC
              GCCATCGCTGACCAGCCCCACGGCTACATCATCATGTTCG
              GTCTGAGCATCCTGATCATGGTTGTGGCCTTGTTCCTAGG
              CATTATCCCTCTGAAAGGTACGCGCAAGTTGACCGATCAG
              ATCGCCTGA
CbsT1 protein MSTTPTQPSSRKQAVYPYLIVLSGIVFTAIPVSLVCSCAGIFFT
Lactobacillus PVSSYFHVPKAAFTGYFSIFSITMVAFLPVAGWLMHRYDLRI
johnsonii     VLTASTVLAGLGCLGMSRSSAMWQFYLCGVVLGIGMPAVL
SEQ ID NO:    YLSVPTLINAWFRKRVGFFIGLCMAFTGIGGVIFNQIGTMIIR
111           SAPDGWRRGYLVFAILILVTTLPFTIFVIRSTPEQMGLHPYGA
              DQEPDAAETATNSAGTGSKDQKSPEPAASTVGMTASQALRS
              PAFWALALFCGLITMNQTIYQFLPSYAASLPSMAAYTGLIAS
              SCMAGQAIGKIILGMVNDGSIVGGLCLGIGGGILGVCLMVAF
              PGLPVLLLLGAFAFGLVYACTTVQTPILVTAVFGSRDYTNIY
              ARIQMVGSLASAFAALFWGAIADQPHGYIIMFGLSILIMVVA
              LFLGHPLKGTRKLTDQIA
cbsT2 from    ATGTCTACTGATGCCGCTACTAAAGATAAAGTAGTAAGC
Lactobacillus AAGGGCTATAAATACTTCATGGTTTTCCTTTGTATGTTAA
Johnsonii     CCCAAGCTATTCCTTATGGAATTGCTCAAAACATTCAGCC
SEQ ID NO:    TTTGTTTATCCACCCTTTAGTTAATACTTTCCACTTTACCT
112           TAGCATCGTACACATTAATTTTTACGTTTGGTGCGGTTTTT
              GCTTCAGTTGCTTCTCCATTTATTGGTAAGGCATTAGAAA
              AAGTTAACTTCCGACTAATGTATTTAATTGGTATTGGTCT
              TTCTGCTATTGCCTACGTAATTTTTGGAATTAGTACAAAA
              CTACCCGGTTTCTATATTGCCGCTATCATTTGTATGGTTGG
              TTCAACCTTTTACTCCGGCCAAGGTGTTCCCTGGGTTATT
              AACCACTGGTTCCCAGCAAAGGGACGTGGGGCTGCCTTA
              GGAATTGCCTTCTGCGGTGGTTCTATTGGTAATATCTTTTT
              ACAACCAGCAACCCAAGCTATTTTAAAACACTACATGAC
              AGGTAATACTAAGACCGGTCATTTAACCTCTATGGCACCA
              TTCTTTATCTTTGCCGTAGCTTTATTAGTAATCGGTGTAAT
              TATCGCCTGCTTCATTAGAACCCCTAAGAAAGACGAAATT
              GTTGTTTCTGATGCAGAACTAGCTGAAAGCAAGAAAGCT
              GAAGCCGCAGCCAAAGCTAAAGAGTTTAAAGGCTGGACT
              AGTAAACAAGTGTTACAAATGAAATGGTTCTGGATTTTCA
              GCCTTGGTTTCTTAATCATTGGTTTAGGCTTAGCTTCTTTA
              AATGAAGACTATGCCGCCTTCCTTGATACTAAGCTTTCTT
              TAACCGATGTTGGTTTAGTTGGGTCAATGTACGGTGTTGG
              TTGTTTAATCGGAAATATTTCTGGTGGTTTCTTATTTGATA
              AATTTGGTACAGCAAAATCAATGACCTATGCTGGTTGTAT
              GTATATTTTATCTATTCTGATGATGATCTTTATTAGCTTCC
              AGCCATATGGTTCATCTATTAGTAAGGCTGCTGGCATTGG
              CTATGCTATCTTTTGCGGCTTAGCTGTATTTAGTTACATGT
              CAGGCCCAGCCTTCATGGCAAAAGACCTCTTTGGTTCAAG
              AGATCAAGGTGTCATGCTTGGATACGTTGGTTTAGCTTAT
              GCAATTGGCTATGCCATTGGTGCTCCACTATTTGGGATTA
              TTAAGGGAGCGGCAAGCTTTACAGTTGCTTGGTACTTTAT
              GATTGCCTTTGTTGCAATTGGTTTTATCATTTTAGTATTTG
              CCGTTATCCAAATTAAGAGATACCAAAAGAAATACATTG
              CAGAGCAAGCAGCAAAAGCTAATGCTAAATAA
CbsT2 protein MSTDAATKDKVVSKGYKYFMVFLCMLTQAIPYGIAQNIQPL
from          FIHPLVNTFHFTLASYTLIFTFGAVFASVASPFIGKALEKVNF
Lactobacillus RLMYLIGIGLSAIAYVIFGISTKLPGFYIAAIICMVGSTFYSGQ
Johnsonii     GVPWVINHWFPAKGRGAALGIAFCGGSIGNIFLQPATQAILK
SEQ ID NO:    HYMTGNTKTGHLTSMAPFFIFAVALLVIGVIIACFIRTPKKDE
113           IVVSDAELAESKKABAAAKAKEFKGWTSKQVLQMKWFWIF
              SLGFLIIGLGLASLNEDYAAFLDTKLSLTDVGLVGSMYGVG
              CLIGNISGGFLFDKFGTAKSMTYAGCMYILSILMMIFISFQPY
              GSSISKAAGIGYAIFCCLAVFSYMSGPAFMAKDLFGSRDQG
              VMLGYVGLAYAIGYAIGAPLFGIIKGAASFTVAWYFMIAFV
              AIGFIILVFAVIQIKRYQKKYIABQAAKANAK
ABCB11 bile   GAATGATGAAAACCGAGGTTGGAAAAGGTTGTGAAACCT
salt exporter TTTAACTCTCCACAGTGGAGTCCATTATTTCCTCTGGCTTC
Homo sapiens  CTCAAATTCATATTCACAGGGTCGTTGGCTGTGGGTTGCA
SEQ ID NO:    ATTACCATGTCTGACTCAGTAATTCTTCGAAGTATAAAGA
114           AATTTGGAGAGGAGAATGATGGTTTTGAGTCAGATAAAT
              CATATAATAATGATAAGAAATCAAGGTTACAAGATGAGA
              AGAAAGGTGATGGCGTTAGAGTTGGCTTCTTTCAATTGTT
              TCGGTTTTCTTCATCAACTGACATTTGGCTGATGTTTGTGG
              GAAGTTTGTGTGCATTTCTCCATGGAATAGCCCAGCCAGG
              CGTGCTACTCATTTTTGGCACAATGACAGATGTTTTTATT
              GACTACGACGTTGAGTTACAAGAACTCCAGATTCCAGGA
              AAAGCATGTGTGAATAACACCATTGTATGGACTAACAGT
              TCCCTCAACCAGAACATGACAAATGGAACACGTTGTGGG
              TTGCTGAACATCGAGAGCGAAATGATCAAATTTGCCAGTT
              ACTATGCTGGAATTGCTGTCGCAGTACTTATCACAGGATA
              TATTCAAATATGCTTTTGGGTCATTGCCGCAGCTCGTCAG
              ATACAGAAAATGAGAAAATTTTACTTTAGGAGAATAATG
              AGAATGGAAATAGGGTGGTTTGACTGCAATTCAGTGGGG
              GAGCTGAATACAAGATTCTCTGATGATATTAATAAAATCA
              ATGATGCCATAGCTGACCAAATGGCCCTTTTCATTCAGCG
              CATGACCTCGACCATCTGTGGTTTCCTGTTGGGATTTTTCA
              GGGGTTGGAAACTGACCTTGGTTATTATTTCTGTCAGCCC
              TCTCATTGGGATTGGAGCAGCCACCATTGGTCTGAGTGTG
              TCCAAGTTTACGGACTATGAGCTGAAGGCCTATGCCAAA
              GCAGGGGTGGTGGCTGATGAAGTCATTTCATCAATGAGA
              ACAGTGGCTGCTTTTGGTGGTGAGAAAAGAGAGGTTGAA
              AGGTATGAGAAAAATCTTGTGTTCGCCCAGCGTTGGGGA
              ATTAGAAAAGGAATAGTGATGGGATTCTTTACTGGATTCG
              TGTGGTGTCTCATCTTTTTGTGTTATGCACTGGCCTTCTGG
              TACGGCTCCACACTTGTCCTGGATGAAGGAGAATATACA
              CCAGGAACCCTTGTCCAGATTTTCCTCAGTGTCATAGTAG
              GAGCTTTAAATCTTGGCAATGCCTCTCCTTGTTTGGAAGC
              CTTTGCAACTGGACGTGCAGCAGCCACCAGCATTTTTGAG
              ACAATAGACAGGAAACCCATCATTGACTGCATGTCAGAA
              GATGGTTACAAGTTGGATCGAATCAAGGGTGAAATTGAA
              TTCCATAATGTGACCTTCCATTATCCTTCCAGACCAGAGG
              TGAAGATTCTAAATGACCTCAACATGGTCATTAAACCAG
              GGGAAATGACAGCTCTGGTAGGACCCAGTGGAGCTGGAA
              AAAGTACAGCACTGCAACTCATTCAGCGATTCTATGACCC
              CTGTGAAGGAATGGTGACCGTGGATGGCCATGACATTCG
              CTCTCTTAACATTCAGTGGCTTAGAGATCAGATTGGGATA
              GTGGAGCAAGAGCCAGTTCTGTTCTCTACCACCATTGCAG
              AAAATATTCGCTATGGCAGAGAAGATGCAACAATGGAAG
              ACATAGTCCAAGCTGCCAAGGAGGCCAATGCCTACAACT
              TCATCATGGACCTGCCACAGCAATTTGACACCCTTGTTGG
              AGAAGGAGGAGGCCAGATGAGTGGTGGCCAGAAACAAA
              GGGTAGCTATCGCCAGAGCCCTCATCCGAAATCCCAAGA
              TTCTGCTTTTGGACATGGCCACCTCAGCTCTGGACAATGA
              GAGTGAAGCCATGGTGCAAGAAGTGCTGAGTAAGATTCA
              GCATGGGCACACAATCATTTCAGTTGCTCATCGCTTGTCT
              ACGGTCAGAGCTGCAGATACCATCATTGGTTTTGAACATG
              GCACTGCAGTGGAAAGAGGGACCCATGAAGAATTACTGG
              AAAGGAAAGGTGTTTACTTCACTCTAGTGACTTTGCAAAG
              CCAGGGAAATCAAGCTCTTAATGAAGAGGACATAAAGGA
              TGCAACTGAAGATGACATGCTTGCGAGGACCTTTAGCAG
              AGGGAGCTACCAGGATAGTTTAAGGGCTTCCATCCGGCA
              ACGCTCCAAGTCTCAGCTTTCTTACCTGGTGCACGAACCT
              CCATTAGCTGTTGTAGATCATAAGTCTACCTATGAAGAAG
              ATAGAAAGGACAAGGACATTCCTGTGCAGGAAGAAGTTG
              AACCTGCCCCAGTTAGGAGGATTCTGAAATTCAGTGCTCC
              AGAATGGCCCTACATGCTGGTAGGGTCTGTGGGTGCAGC
              TGTGAACGGGACAGTCACACCCTTGTATGCCTTTTTATTC
              AGCCAGATTCTTGGGACTTTTTCAATTCCTGATAAAGAGG
              AACAAAGGTCACAGATCAATGGTGTGTGCCTACTTTTTGT
              AGCAATGGGCTGTGTATCTCTTTTCACCCAATTTCTACAG
              GGATATGCCTTTGCTAAATCTGGGGAGCTCCTAACAAAA
              AGGCTACGTAAATTTGGTTTCAGGGCAATGCTGGGGCAA
              GATATTGCCTGGTTTGATGACCTCAGAAATAGCCCTGGAG
              CATTGACAACAAGACTTGCTACAGATGCTTCCCAAGTTCA
              AGGGGCTGCCGGCTCTCAGATCGGGATGATAGTCAATTC
              CTTCACTAACGTCACTGTGGCCATGATCATTGCCTTCTCCT
              TTAGCTGGAAGCTGAGCCTGGTCATCTTGTGCTTCTTCCC
              CTTCTTGGCTTTATCAGGAGCCACACAGACCAGGATGTTG
              ACAGGATTTGCCTCTCGAGATAAGCAGGCCCTGGAGATG
              GTGGGACAGATTACAAATGAAGCCCTCAGTAACATCCGC
              ACTGTTGCTGGAATTGGAAAGGAGAGGCGGTTCATTGAA
              GCACTTGAGACTGAGCTGGAGAAGCCCTTCAAGACAGCC
              ATTCAGAAAGCCAATATTTACGGATTCTGCTTTGCCTTTG
              CCCAGTGCATCATGTTTATTGCGAATTCTGCTTCCTACAG
              ATATGGAGGTTACTTAATCTCCAATGAGGGGCTCCATTTC
              AGCTATGTGTTCAGGGTGATCTCTGCAGTTGTACTGAGTG
              CAACAGCTCTTGGAAGAGCCTTCTCTTACACCCCAAGTTA
              TGCAAAAGCTAAAATATCAGCTGCACGCTTTTTTCAACTG
              CTGGACCGACAACCCCCAATCAGTGTATACAATACTGCA
              GGTGAAAAATGGGACAACTTCCAGGGGAAGATTGATTTT
              GTTGATTGTAAATTTACATATCCTTCTCGACCTGACTCGC
              AAGTTCTGAATGGTCTCTCAGTGTCGATTAGTCCAGGGCA
              GACACTGGCGTTTGTTGGGAGCAGTGGATGTGGCAAAAG
              CACTAGCATTCAGCTGTTGGAACGTTTCTATGATCCTGAT
              CAAGGGAAGGTGATGATAGATGGTCATGACAGCAAAAAA
              GTAAATGTCCAGTTCCTCCGCTCAAACATTGGAATTGTTT
              CCCAGGAACCAGTGTTGTTTGCCTGTAGCATAATGGACAA
              TATCAAGTATGGAGACAACACCAAAGAAATTCCCATGGA
              AAGAGTCATAGCAGCTGCAAAACAGGCTCAGCTGCATGA
              TTTTGTCATGTCACTCCCAGAGAAATATGAAACTAACGTT
              GGGTCCCAGGGGTCTCAACTCTCTAGAGGGGAGAAACAA
              CGCATTGCTATTGCTCGGGCCATTGTACGAGATCCTAAAA
              TCTTGCTACTAGATGAAGCCACTTCTGCCTTAGACACAGA
              AAGTGAAAAGACGGTGCAGGTTGCTCTAGACAAAGCCAG
              AGAGGGTCGGACCTGCATTGTCATTGCCCATCGCTTGTCC
              ACCATCCAGAACGCGGATATCATTGCTGTCATGGCACAG
              GGGGTGGTGATTGAAAAGGGGACCCATGAAGAACTGATG
              GCCCAAAAAGGAGCCTACTACAAACTAGTCACCACTGGA
              TCCCCCATCAGTTGACCCAATGCAAGAATCTCAGACACAC
              ATGACGCACCAGTTACAGGGGTTGTTTTTAAAGAAAAAA
              ACAATCCCAGCAGGAGGGATTGCTGGGATTGTTTTTTCTT
              TAAAGAAGAATGTTAATATTTTACTTTTACAGTCATTTTC
              CTACATCGGAATCCAAGCTAATTTCTAATGGCCTTCCATA
              ATAATTCTGCTTTAGATGTGTATACAGAAAATGAAAGAA
              ACTAGGGTCCATATGAGGGAAAACCCAATGTCAAGTGGC
              AGCTCAGCCACCACTCAGTGCTTCTCTGTGCAGGAGCCAG
              TCCTGATTAATATGTGGGAATTAGTGAGACATCAGGGAG
              TAAGTGACACTTTGAACTCCTCAAGGGCAGAGAACTGTCT
              TTCATTTTTGAACCCTCGGTGTACACAGAGGCGGGTCTAT
              AACAGGCAATCAACAAACGTTTCTTGAGCTAGACCAAGG
              TCAGATTTGAAAAGAACAGAAGGACTGAAGACCAGCTGT
              GTTTCTTAACTAAATTTGTCTTTCAAGTGAAACCAGCTTC
              CTTCATCTCTAAGGCTAAGGATAGGGAAAGGGTGGATGC
              TCTCAGGCTGAGGGAGGCAGAAAGGGAAAGTATTAGCAT
              GAGCTTTCCAGTTAGGGCTGTTGATTTATGCTTTAACTTC
              AGAGTGAGTGTAGGGGTGGTGATGCT
ABCB11 bile   MSDSVILRSIKKFGEENDGFESDKSYNNDKKSRLQDEKKGD
salt exporter GVRVGFFQLFRFSSSTDIWLMFVGSLCAFLHGIAQPGVLLIF
protein Homo  GTMTDVFIDYDVELQELQIPGKACVNNTIVWTNSSLNQNMT
sapiens       NGTRCGLLNIESEMIKFASYYAGIAVAVLITGYIQICFWVIAA
SEQ ID NO:    ARQIQKMRKFYFRRIMRMEIGWFDCNSVGELNTRFSDDINKI
115           NDAIADQMALFIQRMTSTICGFLLGFFRGWKLTLVIISVSPLI
              GIGAATIGLSVSKFTDYELKAYAKAGVVADEVISSMRTVAA
              FGGEKREVERYEKNLVFAQRWGIRKGIVMGFFTGFVWCLIF
              LCYALAFWYGSTLVLDEGEYTPGTLVQIFLSVIVGALNLGN
              ASPCLEAFATGRAAATSIFETIDRKPIIDCMSEDGYKLDRIKG
              EIEFHNVTFHYPSRPEVKILNDLNMVIKPGEMTALVGPSGAG
              KSTALQLIQRFYDPCEGMVTVDGHDIRSLNIQWLRDQIGIVE
              QEPVLFSTTIAENIRYGREDATMEDIVQAAKEANAYNFIMDL
              PQQFDTLVGEGGGQMSGGQKQRVAIARALIRNPKILLLDMA
              TSALDNESEAMVQEVLSKIQHGHTIISVAHRLSTVRAADTIIG
              FEHGTAVERGTHEELLERKGVYFTLVTLQSQGNQALNEEDI
              KDATEDDMLARTFSRGSYQDSLRASIRQRSKSQLSYLVHEPP
              LAVVDHKSTYEEDRKDKDIPVQEEVEPAPVRRILKFSAPEWP
              YMLVGSVGAAVNGTVTPLYAFLFSQILGTFSIPDKEEQRSQI
              NGVCLLFVAMGCVSLFTQFLQGYAFAKSGELLTKRLRKFGF
              RAMLGQDIAWFDDLRNSPGALTTRLATDASQVQGAAGSQI
              GMIVNSFTNVTVAMIIAFSFSWKLSLVILCFFPFLALSGATQT
              RMLTGFASRDKQALEMVGQITNEALSNIRTVAGIGKERRFIE
              ALETELEKPFKTAIQKANIYGFCFAFAQCIMFIANSASYRYG
              GYLISNEGLHFSYVFRVISAVVLSATALGRAFSYTPSYAKAK
              ISAARFFQLLDRQPPISVYNTAGEKWDNFQGKIDFVDCKFTY
              PSRPDSQVLNGLSVSISPGQTLAFVGSSGCGKSTSIQLLERFY
              DPDQGKVMIDGHDSKKVNVQFLRSNIGIVSQEPVLFACSIM
              DNIKYGDNTKEIPMERVIAAAKQAQLHDFVMSLPEKYETNV
              GSQGSQLSRGEKQRIAIARAIVRDPKILLLDEATSALDTESEK
              TVQVALDKAREGRTCIVIAHRLSTIQNADIIAVMAQGVVIEK
              GTHEELMAQKGAYYKLVTTGSPIS
Streptococcus MEGRTVFVIAHRLSTIVNSDVILVMDHGRIIKRGDHDTLMEQ
thermophilus  GGTYYRLYTGSLEID
Msba
subfamily
ABC
transporter
ATP-binding
protein
STH8232_1633
SEQ ID NO:
116
Nostoc spp.   ATGTGGGGGAAACAAAGACAAAGAATCGCCATTGCACGA
As11293 ABC   GGGGGTTTTAAGAATTTGCAGGTTTTGATTTTAGATAAAG
transporter   CAACCTCGGCATTGGATAATAAAACAGAAGCAGCTATTG
gene          AGCGATCGCTGGTGTTGACTGTTGACCAATGA
SEQ ID NO:
117
Nostoc spp.   MWGKQRQRIAIARGGFKNLQVLILDKATSALDNKTEAMER
As11293 ABC   SINLTVDQ.
transporter
protein
SEQ ID NO:
118
Neisseria     ATGAATATCCTCAGTAAAATCAGCAGCTTTATCGGAAAA
meningitides  ACATTTTCCCTCTGGGCCGCGCTCTTTGCCGCCGCCGCTTT
(MC58)        TTTCGCGCCCGACACCTTCAAATGGGCGGGGCCTTATATT
ASBTNN4 bile  CCTTGGCTGTTGGGCATTATTATGTTCGGTATGGGTTTGA
acid sodium   CGCTCAAACCTTCCGACTTCGATATTTTGTTCAAACATCC
symporter     CAAAGTCGTCATCATCGGCGTAATCGCACAATTCGCCATT
(NMB0705)     ATGCCGGCAACCGCCTGGCTGCTGTCCAAACTGTTGAACC
SEQ ID NO:    TGCCTGCCGAAATCGCGGTCGGCGTGATTTTGGTCGGCTG
119           CTGCCCGGGCGGTACGGCTTCCAATGTGATGACCTATCTG
              GCGCGTGGCAATGTGGCTTTGTCGGTTGCCGTTACGTCTG
              TTTCCACCCTGATTTCCCCATTGCTGCTCCCGCCATCTTC
              TTTCCACCCTGATTTCCCCATTGCTGACTCCCGCCATCTTC
              CTGATGCTTGCCGGCGAAATGCTGGAAATCCAAGCGGCC
              GGTATGTTGATGTCCATCGTCAAAATGGTTTTGCTCCCCA
              TTGTTTTGGGTTTGATTGTCCATAAGGTTTTGGGCAGTAA
              AACCGAAAAGCTGACCGATGCGCTGCCGCTGGTTTCCGTT
              GCCGCCATCGTGCTGATTATCGGCGCGGTTGTTGGGGCAA
              GCAAAGGCAAGATTATGGAAAGCGGCCTTGCTGATTTTTG
              CGGTTGTCGTACTCCACAACGGCATCGGCTACCTGCTCGG
              CTTCTTTGCCGCCAAATGGACCGGCCTGCCTTATGATGCA
              CAAAAAACGCTGACCATCGAAGTCGGTATGCAAAACTCG
              GGCCTGGCCGCCGCGCTTGCCGCCGCACACTTTGCCGCCG
              CGCCGGTCGTTGCCGTTCCGGGCGCATTGTTCAGCGTGTG
              GCACAATATCTCCGGCTCGCTGCTGGCAACTTATTGGGCG
              GCCAAAGCCGGTAAACATAAAAAACCCTAA
Neisseria     MNILSKISSFIGKTFSLWAALFAAAAFFAPDTFKWAGPYIPW
meningitides  LLGIIMFGMGLTLKPSDFDILFKHPKVVIIGVIAQFAIMPATA
(MC58)        WLLSKLLNLPAEIAVGVILVGCCPGGTASNVMTYLARGNVA
ASBTNm bile   LSVAVTSVSTLISPLLTPAIFLMLAGEMLEIQAAGMLMSIVK
acid sodium   MVLLPIVLGLIVHKVLGSKTEKLTDALPLVSVAAIVLIIGAVV
symporter     GASKGKIMESGLLIFAVVVLHNGIGYLLGFFAAKWTGLPYD
protein       AQKTLTIEVGMQNSGLAAALAAAHFAAAPVVAVPGALFSV
SEQ ID NO:    WHNISGSLLATYWAAKAGKHKKPGSENLYFQ
120

In one embodiment, the bile salt transporter is the bile salt importer CbsT1. In one embodiment, the cbsT1 gene has at least about 80% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 90% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 95% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 110. In another embodiment, the cbsT1 gene comprises the sequence of SEQ ID NO: 110. In yet another embodiment the cbsT1 gene consists of the sequence of SEQ ID NO: 110.

In one embodiment, the bile salt transporter is the bile salt importer CbsT2. In one embodiment, the cbsT2 gene has at least about 80% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 90% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 95% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 112. In another embodiment, the cbsT2 gene comprises the sequence of SEQ ID NO: 112. In yet another embodiment the cbsT2 gene consists of the sequence of SEQ ID NO: 112.

In one embodiment, the bile acid transporter is the bile acid sodium symporter ASBT_NM. In one embodiment, the NMB0705 gene of Neisseria meningitides has at least about 80% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 90% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 95% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 117. In another embodiment, the NMB0705 gene comprises the sequence of SEQ ID NO: 117. In yet another embodiment the NMB0705 gene consists of the sequence of SEQ ID NO: 117.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120.

In some embodiments, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to the first promoter. In other embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to a second promoter. In one embodiment, the gene encoding a transporter of the bile salt is directly operably linked to the second promoter. In another embodiment, the gene encoding a transporter of the bile salt is indirectly operably linked to the second promoter.

In some embodiments, expression of a gene encoding a transporter of a bile salt is controlled by a different promoter than the promoter that controls expression of the gene encoding the bile salt hydrolase enzyme. In some embodiments, expression of the gene encoding a transporter of a bile salt is controlled by the same promoter that controls expression of the bile salt hydrolase enzyme. In some embodiments, a gene encoding a transporter of a bile salt and the bile salt hydrolase enzyme are divergently transcribed from a promoter region. In some embodiments, expression of each of genes encoding the gene encoding a transporter of a bile salt and the gene encoding the bile salt hydrolase enzyme is controlled by different promoters.

In one embodiment, the gene encoding a transporter of a bile salt is not operably linked with its natural promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by an inducible promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a promoter that is stronger than its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a constitutive promoter.

In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra.

In one embodiment, the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of the gene encoding a transporter of a bile salt from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the at least one native gene encoding the transporter of a bile salt in the bacterial cell is not modified, and one or more additional copies of the native transporter of a bile salt are inserted into the genome. In one embodiment, the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the bile salt hydrolase enzyme, or a constitutive promoter. In alternate embodiments, the at least one native gene encoding the transporter is not modified, and one or more additional copies of the transporter from a different bacterial species is inserted into the genome of the bacterial cell. In one embodiment, the one or more additional copies of the transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the gene encoding the bile salt hydrolase enzyme, or a constitutive promoter.

In one embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 10% more bile salt into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more bile salt into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import two-fold more bile salt into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more bile salt into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

Exporters of Bile Salts

The export of bile salts is mediated by proteins well known to those of skill in the art. For example, the ATP-binding cassette, sub-family B member 11 (ABCB11, also called BSEP or “bile salt export pump”) is responsible for the export of taurochoate and other cholate conjugates from hepatocytes to the bile in mammals, and mutations in this gene have been associated with progressive familial intrahepatic cholestasis type 2 (PFIC2) and hepatocellular carcinoma (see Strautnieks et al., Nature Genetics, 20(3):233-238, 1998; Knisely et al., Hepatology, 44(2):478-486, 2006; and Ho et al., Pharmacogenet. Genomics, 20(1):45-57, 2010; SEQ ID NO: 113 and SEQ ID NO:114). In bacteria, Streptococcus thermophilus comprises a bile salt export pump (Msba subfamily ABC transporter ATP-binding protein; accession F8LYG6; SEQ ID NO: 116), and Nostoc spp. are known to comprise a bile salt export pump (As11293; accession Q8YXC2; SEQ ID NO: 117 and SEQ ID NO: 118). Multiple other bile salt exporters are known in the art.

Thus, in one embodiment of the invention, when the recombinant bacterial cell comprises an endogenous bile salt exporter gene, the recombinant bacterial cells may comprise a genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cell comprises a genetic modification that reduces export of one or more bile salts from the bacterial cell and a heterologous gene encoding a bile salt catabolism enzyme. When the recombinant bacterial cells comprise a genetic modification that reduces export of a bile salt, the bacterial cells retain more bile salts in the bacterial cell than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a genetic modification that reduces export of a bile salt may be used to retain more bile salts in the bacterial cell so that any bile salt catabolism enzyme expressed in the organism can catabolize the bile salt(s) to treat diseases associated with bile salts, including cardiovascular disease. In one embodiment, the recombinant bacteria further comprise a heterologous gene encoding a transporter of one or more bile salts.

In one embodiment, the recombinant bacterial cell comprises a genetic modification in a gene encoding a bile salt exporter wherein said bile salt exporter comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a bile salt exporter gene disclosed herein. In one embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 115. In another embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 117.

In one embodiment, the genetic modification reduces export of a bile salt from the bacterial cell. In one embodiment, the bacterial cell is from a bacterial genus or species that includes but is not limited to, Streptococcus thermophilus or Nostoc spp.

In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in an exporter having reduced activity as compared to a wild-type exporter protein. In one embodiment, the activity of the exporter is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the exporter is reduced at least two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in an exporter having no activity, i.e., results in an exporter which cannot export one or more bile salts from the bacterial cell.

It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of the exporter of an amino acid. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; U.S. Pat. No. 6,586,182; U.S. Pat. No. 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462; Carter, 1986, “Site-directed mutagenesis,” Biochem. J., 237:1-7; and Minshull, et al., 1999, “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290. For example, the lambda red system can be used to knock-out genes in E. coli (see, for example, Datta et al., Gene, 379:109-115 (2006)).

The term “inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). The term “inactivated” encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene's coding sequence. The term “knockout” refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome.

Assays for testing the activity of an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, export of one or more bile salts may be determined using the methods described by Telbisz and Homolya, Expert Opinion Ther. Targets, 1-14, 2015, the entire contents of which are expressly incorporated herein by reference.

In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in decreased expression of the exporter gene. In one embodiment, exporter gene expression is reduced by about 50%, 75%, or 100%. In another embodiment, exporter gene expression is reduced about two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation completely inhibits expression of the exporter gene.

Assays for testing the level of expression of a gene, such as an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, reverse-transcriptase polymerase chain reaction may be used to detect the level of mRNA expression of a gene. Alternatively, Western blots using antibodies directed against a protein may be used to determine the level of expression of the protein.

In another embodiment, the genetic modification is an overexpression of a repressor of an exporter of one or more bile salts. In one embodiment, the overexpression of the repressor of the exporter is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the exporter is caused by the insertion of an inducible promoter in front of the repressor so that the expression of the repressor can be induced. Inducible promoters are described in more detail herein.

In one embodiment, the recombinant bacterial cells described herein comprise at least one genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise two genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise three genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise four genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise five genetic modifications that reduce export of one or more bile salts from the bacterial cell. GLP-2

In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-2 or proglucagon. Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function (Yazbeck et al., 2009). Obesity is associated with systemic inflammation and intestinal permeability, and commensal bacteria that produce GLP-2 may ameliorate those symptoms of the metabolic disease (Musso et al., 2010). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-2 or proglucagon in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 121 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 121 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 in low-oxygen conditions.

TABLE 12A
SEQ ID NO: 121 GLP-2
SEQ ID NO: 121
HADGSFSDEMNTILDNLAARDFINWLIQTKITD

In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 analogs, including but not limited to, Gattex and teduglutide. Teduglutide is a protease resistan analog of GLP-2. It is made up of 33 amino acids and differs from GLP-2 by one amino acid (alanine is substituted by glycine). The significance of this substitution is that teduglutide is longer acting than endogenous GLP-2 as it is more resistant to proteolysis from dipeptidyl peptidase-4.

TABLE 12B
SEQ ID NO: 122 Teduglutide
SEQ ID NO: 122
HGDGSFSDEMNTILDNLAARDFINWLIQTKITD

In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 122 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 122 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide in low-oxygen conditions. In any of these embodiments the gene sequence encoding GLP-2 or GLP-2 analog may be operably linked to any of the indicuible promoters described herein. In any of these embodiments, the gene sequence encoding GLP-2 or GLP-2 analog may be operably linked to apromoter that it is not naturallyt linked to in nature.

Tryptophan and Metabolites

1-Tryptophan (TRP) is one of the nine essential amino acids and is the least abundant of all 21 dietary amino acids in human beings. Dietary TRP is transported from the digestive tract through the portal vein to the liver where it is used for the synthesis of proteins. The distinguishing structural characteristic of TRP is that it contains an indole functional group. Apart from protein synthesis, TRP is used in the generation of products such as serotonin, melatonin, tryptamine, indole and other indole metabolites, and kynurenine pathway metabolites (KP, collectively called the kynurenines). TRP and its catabolites have well characterized immunosuppressive and disease tolerance functions, and contribute to immune privileged sites such as eyes, brain, placenta, and testes. The kynurenine pathway represents >95% of TRP- catabolizing pathways and is now established as a key regulator of innate and adaptive immunity through its involvement in cancer, autoimmunity, infection, and gastrointestinal health and gut barrier integrity, and other inflammatory metabolic disorders.

Several KP Pathway metabolites, most notably kynurenine, have been shown to be activating ligands for the arylcarbon receptor (AhR; also known as dioxin receptor). Kynurenine (KYN) was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival.

In the gut, the kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism. Tryptophan may be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell responses and promotion of Treg cells.

More recently, additional tryptophan metabolites, collectively termed “indoles”, herein, also have been shown to function as AhR agonists. The metabolites include for example, indole-3 aldehyde, indole-3 acetate, indole-3 propionic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc., and tryptamine (are, see e.g., Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 35A and FIG. 35B and elsewhere herein, and Lama et al., Nat Med. 2016 Jun; 22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands). The majority of these metabolites are generated by the microbiota; some are generated by the human host and/or taken up from the diet.

Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in addition to kynurenine, other tryptophan metabolites, e.g., indoles (described in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, tryptamine, and kynurenic acide (KYNA) have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-KB subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT associated transcription of IL-6, which induced autocrine activation of IDO1 via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated immunosuppression enables the immune escape of tumor cells.

More recently, some indole metabolites, including but not limited to indole 3 propionic acid, have been shown to exert their effect through Pregnane X receptor (PXR), which also thought to play a key role as an essential regulator of intestinal barrier function.

Kynurenine Pathway

Kynurenine, IDO, and TDO

The rate-limiting conversion of tryptophan to kynurenine (KYN) may be mediated by either of two forms of indoleamine 2, 3-dioxygenase, IDO1 expressed ubiquitously, IDO2 expressed in kidneys, epididymis, testis, and liver or by tryptophan 2,3-dioxygenase (TDO) expressed in the liver and brain.

The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of TRP:KYN.

In one embodiment, the genetically engineered bacteria comprise any suitable gene or genes for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise one or more of the following: a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions. In some embodiments the genetically engineered bacteria secrete an enzyme which produces kynurenine.

Post-Kynurenine Metabolism

As shown in FIG. 32, kynurenine is further metabolized along the two distinct routes competing for kynurenine as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway.

Kynurenic Acid, Xanthurenic Acid, Anthranillic Acid

Kynurenine is further metabolized along the two distinct routes competing for KYN as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway. Along one arm, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA). KYNA is generated by kynurenine aminotransferases (KAT I, II, III) and can also bind AHR and GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMDA)- receptors.

The major nerve supply to the gut is also activated the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, increasing or decreasing kynurenic acid levels may be beneficial to optimally regulate gut motility or gut inflammation.

KYNA also has signaling functions through activation of its recently identified receptor, GPR35. GPR35 is predominantly detected in immune cells in the gastrointestinal tract, and might be involved in nociceptive perception. KYNA might have an anti-inflammatory effect by inhibition of lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha secretion in peripheral blood mononuclear cells.

Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, presumably due to chronic stress or the low-grade inflammation that are prominent risk factors for diabetes. Thermochemical and kinetic data show that KYNA and XA are the best free- radical scavengers from the eight tested TRP metabolites, suggesting that the production is a regulatory mechanism to attenuate damage by the inflammation-induced production of reactive oxygen species, e.g., in type two diabetes.

The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid, e.g., from kynurenine through a circuit comprising gene(s) or gene sequence(s) compring kynurenine--oxoglutarate transaminase or an equivalent thereof. In some embodiments, the genetically engineered bacteria comprising gene(s) or gene sequence(s) encoding kynurenine--oxoglutarate transaminase.

In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions. In some embodiments, the genetically engineered bacteria secrete an enzyme for the production of kynurenic acid.

In other embodiments, the genetically engineered bacteria are capable of reducing levels of kynurenic acid, e.g., though overexpression of enzymes catabolizing kynurenic acid described herein.

The KYN-nicotinamide adenine dinucleotide Pathway

The major enzymes of the KYN-NAD pathway are KYN-3-monooxygenase and kynureninase. Among more than 30 intermediate metabolites (collectively named “kynurenines”) are NMDA agonists (quinolinic and picolinic acids) and free radical generators (3-hydroxykynurenine and 3-hydroxyanthranilic acids). One metabolite, xanthurenic acid, reacts with insulin with formation of a complex indistinguishable from insulin. Quinolinic acid (a glutamate receptor agonist) and picolinic acids stimulate inducible nitric oxide synthase (iNOS and together with 3- hydroxykynurenine and 3-hydroxyanthranilic acids might increase lipid peroxidation, and trigger an arachidonic acid cascade resulting in the increased production of inflammatory factors. As such a means to downregulate such KP metabolites is beneficial, e.g., in the treatment of inflammatory metablic diseases, e.g., T2DM and others described herein.

Further, Anthranilic and xanthurenic acid can act as antioxidants in certain chemical environments.

Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of metablic diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning tryptophan and tryptophan metabolite levels, e.g., KP metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites, e.g., KP metabolites, and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.

In certain embodiments, the genetically engineered bacteria comprise one or more genes(s) or gene cassettes, which can synthesize tryptophan and/or one or more of its metabolites, e.g., KP metablites, thereby modulating local and/or systemic concentrations and or ratios of tryptophan and/or one or more of its metabolites.

In some embodiments, the genetically engineered bacteria modulate the inflammatory status, influence immunosuppression, disease tolerance, gut barrier function, satiety.

Other Indole Tryptophan Metabolites

In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles; Nature Scientific Reoports 5:12689).

In the gastronintestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372-385, August 22, 2013). AHR is essential for IL-22-production in the intestinal lamina propria (Lee et al., Nature Immunology 13, 144-151 (2012); AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch).

Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of IL-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

Additionally, indole metabolites have been suggested to be beneficial in the treatment of metabolic disease, such as type2 diabetes. For example, in addition to its enhancement of the gut barrier function, indole has been found to promote GLP-1 secretion by intestinal enteroendocrine cells, i.e, indole inhibits voltage-gated K+channels, and changes the action potential properties of L cells, ultimately triggering GLP-1 secretion (Chimerel C, et a., (2014) Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells. Cell Rep 9:1202-1208).

Table 13 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure. Thus, in some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes for the production of one or more metabolites listed in Table 13.

TABLE 13
Indole Tryptophan Metabolites
Origin         Compound
Exogenous      2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
Dietary        Indole-3-carbinol (I3C)
Dietary        Indole-3-acetonitrile (I3ACN)
Dietary        3.3′-Diindolylmethane (DIM)
Dietary        2-(indol-3-ylmethyl)-3.3′-diindolylmethane (Ltr-1)
Dietary        Indolo(3,2-b)carbazole (ICZ)
Dietary        2-(1′H-indole-3′-carbony)-thiazole-4-carboxylic
               acid methyl ester (ITE)
Microbial      Indole
Microbial      Indole-3-acetic acid (IAA)
Microbial      Indole-3-aldehyde (IAId)
Microbial      Tryptamine
Microbial      3-methyl-indole (Skatole)
Yeast          Tryptanthrin
Microbial/Host Indigo
Metabolism
Microbial/Host Indirubin
Metabolism
Microbial/Host Indoxyl-3-sulfate (I3S)
Metabolism
Host           Kynurenine (Kyn)
Metabolism
Host           Kynurenic acid (KA)
Metabolism
Host           Xanthurenic acid
Metabolism
Host           Cinnabarinic acid (CA)
Metabolism
UV-Light       6-formylindolo(3,2-b)carbazole (FICZ)
Oxidation
Microbial
metabolism

In addition, some indole metabolites may exert their effect through Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nr1i2-/-) mice showed a distinctly “leaky”gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo.

As a result of PXR agonism, indole metabolite levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. In other words, low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinal barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.

In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 43 and FIG. 44 and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes selected from TrpDH: tryptophan dehydrogenase (e.g., from from Nostoc punctiforme NIES-2108); FldH1/F1dH2: indole-3-lactate dehydrogenase (e.g., from Clostridium sporogenes); FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase (e.g., from Clostridium sporogenes); FldBC: indole-3-lactate dehydratase, (e.g., from Clostridium sporogenes); FldD: indole-3-acrylyl-CoA reductase (e.g., from Clostridium sporogenes); AcuI: acrylyl-CoA reductase (e.g., from Rhodobacter sphaeroides); 1pdC: Indole-3-pyruvate decarboxylase (e.g., from Enterobacter cloacae); 1ad1: Indole-3-acetaldehyde dehydrogenase (e.g., from Ustilago maydis); and Tdc: Tryptophan decarboxylase (e.g., from Catharanthus roseus or from Clostridium sporogenes). In some embodiments, the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the production of one or more of the following: indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA).

Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and H⁺. Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (F1dA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA).

Although microbial degradation of tryptophan to indole-3-propionate has been shown in a number of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr 1; 107(3):283-8), to date, the bacterial entire biosynthetic pathway from tryptophan to IPA is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3-pyruvate, indole-3-lactate, and indole-3-acrylate to indole-3-propionate (O′Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys. 1968 Sep 20; 127(1):361-9). Two enzymes that have been purified from C. sporogenes are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968 Apr.; 14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an aminotransferase to indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus tryptophan is also catabolized to indole-3-lactate through successive transamination and dehydrogenation (see, e.g., Tryptophan catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein).

L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indo1-3y1)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3y1) pyruvate and NADH and H+ to indole-3 lactate and NAD+.

In some embodiments, the engineered bacteria comprise gene sequence encoding one or more enzymes selected from tryptophan transaminase (e.g., from C. sporogenes) and/or indole-3-lactate dehydrogenase (e.g., from C. sporogenes), and/or indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus.

In other embodiments, the engineered bacteria comprise IPA-producing circuits comprising enzymes depicted and described in FIG. 43 and FIG. 44 and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence encoding one or more enzymes shown in FIG. 43 and FIG.44.

Methoxyindole pathway, Serotonin and Melatonin

The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tphl or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5-HTP). Subsequently, 5-HTP undergoes decarboxylation to serotonin.

The majority (95%-98%) of total body serotonin is found in the gut (Berger et al., 2009). Peripheral serotonin acts autonomously on many cells, tissues, and organs, including the cardiovascular, gastrointestinal, hematopoietic, and immune systems as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin functions as a ligand for any of 15 membrane-bound mostly G protein-coupled serotonin receptors (5-HTRs) that are involved in various signal transduction pathways in both CNS and periphery. Intestinal serotonin is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. Gut microbiota are interconnected with serotonin signaling and are for example capable of increasing serotonin levels through host serotonin production (Jano et al., Cell. 2015 Apr 9; 161(2):264-76. doi: 10.1016/j.ce11.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis).

Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders and obesity related disorders, such as type 2 diabetes. In mice that lacked the 5-1-iT2C receptor. insulin resistance and development of type 2 diabetes was observed and they later overate and became obese, and 5-HT2C receptor agonists improve blood glucose tolerance.

In some embodiments, the engineered bacteria comprise gene sequence encoding one or more tryptophan hydroxylase genes (Tph1 or Tph2). In some embodiments, the engineered bacteria further comprise gene sequence for decarboxylating 5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for the production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered bacteria comprise gene sequence for the production of seratonin.

In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g., decrease or increase serotonin levels, e.g, in the gut and in the circulation. In certain embodiments, the genetically engineered bacteria influence serotonin synthesis, release, and/or degradation. In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to improve gut barrier function, modulate the inflammatory status, improve glucose tolerance, reduce insulin resistance or otherwise ameliorate symptoms of a metabolic disease and/or an gastrointestinal disorder or inflammatory disorder. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In some embodiments, the genetically engineered bacteria release serotonin into the environment, e.g., the gut. In some embodiments, the genetically engineered modulate or influence serotonin levels produced by the host. In some embodiments, the genetically engineered bacteria counteract microbiota which are responsible for altered serotonin function in many metabolic diseases.

In some embodiments, the genetically engineered bacteria comprise gene sequence encoding tryptophan hydroxylase (TpH (land/or2)) and/or 1-amino acid decarboxylase, e.g. for the treatment of constipation-associated metablic disorders. In some embodiments, the genetically engineered bacteria comprise genetic cassettes which allow trptophan uptake and catalysis, reducing trptophan availability for serotonin synthesis (serotonin depletion). In some embodiments, the genetically engineered bacteria comprise cassettes which promote serotonin uptake from the environment, e.g., the gut, and serotonin catalysis.

Additionally, serotonin also functions a substrate for melatonin biosynthesis. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. It has been postulated that melatonin may have a role in glucose metabolism, and several lines of evidence suggest that low melatonin secretion or reduced melatonin signaling can impair insulin sensitivity and lead to type 2 diabetes. For example, Loss-of-function mutations in the melatonin receptor are associated with insulin resistance and type 2 diabetes and McMullan et al observed that lower melatonin secretion was iassociated with a higher risk of developing type 2 diabetes. (see, e.g., McMullan et al., Melatonin secretion and the incidence of type 2 diabetes JAMA. 2013 Apr. 3; 309(13): 1388-1396).

In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d-erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As a non-limiting example, the cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). “Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources”. Journal of Chemical Biology 5 (1): 5-17. doi:10.1007/s12154-011-0064-8.

In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase (TPH), hydroxyl-O-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic-amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents.

Tryptophan and Tryptophan Metabolite Circuits

Decreasing Exogenous Tryptophan

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria.

The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in E. coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole, et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).

In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. In one embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli tnaB gene.

In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or Corynebacterium glutamicum. In some embodiments, the bacterial species is Escherichia coli. In some embodiments, the bacterial species is Escherichia coli strain Nissle.

Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein.

In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine metabolites, or indole tryptophan metabolites as shown in Table 13.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein. In some embodiments, the gene sequences(s) are driven by an inducible promoter. In some embodiments, the gene sequences(s) are driven by a constitutive promoter.

Increasing Kynurenine

In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37E and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine-oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido 1 and/or tdo2 and/or bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido 1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.

In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation, and/or a metabolic disorder. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with metabolic disorders, such as liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein.

In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Increasing Tryptophan

In some embodiments, the genetically engineered microorganisms of the present disclosure are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in FIG. 36(A-D), FIG. 37A.

In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of E. coli . (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of B. subtilis. (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis.

Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes.

In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 10). Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved (see, e.g., FIG. 40A and FIG. 40B, FIG. 36C, FIG. 36D.

In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 15).

In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.

In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 15.

The inner membrane protein YddG of Escherichia coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, liver damage, and.or metabolic disease, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Table 14 lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.

TABLE 14
Tryptophan Synthesis Cassette Sequences
Description   Sequence
Tet-regulated taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggact
Tryptophan    gcaccttggtgatcaaataattcgatagatgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct
operon        tattagcgacttgatgacttgatatccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata
SEQ ID NO:    atgcattactagtgaaaaaccttgaggcataaaaaggctaattgattttcgagagtttcatactgatttctgtagg
123           ccgtgtacctaaatgtacttttgaccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa
              aaaatatgccagattcccatctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg
              tcgagcaaagcccgcttattattacatgccaatacaatgtaggctgactacacctagatctgggcgagtttacg
              ggttgttaaaccttcgattccgacctcattaagcagactaatgcgctgttaatcactttacttttatctaatctagaca
              tcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtg
              aactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaactgct
              aacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccggcaacg
              ctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc
              gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactg
              gataacgccagcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcacccgcctgtca
              gtccactgctggatgaagacgcccgcttatgctcccatcggtattgacgctaccgcttattacagaatctgaga
              atgtaccgaaggaagaacgagaagcaatgacttcggcggcctgactcttatgaccagtggcgggatttgaaa
              atttaccgcaactgtcagcggaaaatagctgccctgatactgatttatctcgctgaaacgctgatggtgattgac
              catcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgc
              tcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtaccgtgccgcatat
              gcgagtgaatgtaaccagagcgatgaagagacggtggtgtagtgcgatgagcaaaaagcgattcgcgccg
              gagaaattaccaggtggtgccatctcgccgtactctctgccctgcccgtcaccgctggcagcctattacgtgct
              gaaaaagagtaatcccagcccgtacatgatatatgcaggataatgatttcaccctgatggcgcgtcgccggaa
              agacgctcaagtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtc
              gtcgtgccgatggacgctggacagagacctcgacagccgcatcgaactggagatgcgtaccgatcataaag
              agctactgaacatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagccgc
              tacgtcgccgatctcaccaaagagaccgttactcttacgtgatgcacctagtctcccgcgagaggtgagctgc
              gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagcggtgcaccgaaagt
              acgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcggcagctacggcggcgcggtaggttatat
              accgcgcatggcgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgc
              aagccggtgctggcgtagtccagattctgaccgcagtcggaagccgacgaaactcgtaataaagcccgcgc
              tgtactgcgcgctattgccaccgcgcatcatgcacaggagacgactaatggctgacattctgctgctcgataat
              atcgactcattacgtacaacctggcagatcagagcgcagcaatggtcataacgtggtgatttaccgcaaccata
              accggcgcagaccttaattgaacgcctggcgacgatgagcaatccggtgctgatgctactcctggccccggt
              gtgccgagcgaagccggagtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatag
              cctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcacacggtaaa
              gcgtcgagcattgaacatgacggtcaggcgatgatgccggattaacaaacccgctgccagtggcgcgttatc
              actcgctggaggcagtaacattccggccggataaccatcaacgcccatataatggcatggtgatggcggtgc
              gtcacgatgcagatcgcgtagtggattccagaccatccggaatccattcttactacccagggcgctcgcctgct
              ggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattctggaaaaa
              ctgtatcaggcacagacgcttagccaacaagaaagccaccagctgattcagcggtggtacgtggcgagctga
              agccggaacaactggcggcggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccgg
              ggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgatgccgatatcgtcggt
              actggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctga
              aagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgacggtat
              taatatgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgatcctattgcgccgaa
              gtatcacaccggattccgccatgcgatgccggacgccagcaactgaaaacccgcactctgacaacgtgctg
              ggaccattgattaacccggcgcatccgccgctggcgctaattggtgatatagtccggaactggtgctgccgatt
              gccgaaaccagcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggcgggatggatgaagatc
              attacacgcgccgacaatcgagccgaactacatgacggcgaaattaagagctatcaattgaccgctgaagatt
              ttggcctgacaccctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacac
              gcagttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgaaatgcgcct
              gcatggccatgaagatctgcaagccaatgcgcaaaccgacttgaggtactgcgcagtggaccgcttacgaca
              gagtcaccgcactggcggcacgagggtaaatgatgcaaaccgattagcgaaaatcgtcgcagacaaggcg
              atttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga
              cattatatgatgcacttcagggcgcacgcacggcgatattctggagtgtaaaaaagcgtcgccgtcaaaaggc
              gtgatccgtgatgatacgatccggcacgcattgccgccatttataaacattacgatcggcaatacagtgctgac
              tgatgagaaatattacaggggagattgatacctccccatcgtcagccaaatcgccccgcagccgatatatgta
              aagacacattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgattcag
              tactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagt
              cagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgaggcatcaacaaccgcgatct
              gcgcgatagtcgattgatctcaaccgtacccgcgagcttgcgccgaaactggggcacaacgtgacggtaatc
              agcgaatccggcatcaatacttacgctcaggtgcgcgagttaagccacttcgctaacggctactgattggacg
              gcgttgatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcct
              gacacgtgggcaagatgctaaagcagatatgacgcgggcgcgatttacggtgggttgatttttgttgcgacat
              caccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgtt
              ccgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggt
              aatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag
              tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggga
              gcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttaggc
              gcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaac
              cgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaatga
              caacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgcca
              gctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaaact
              atgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatctga
              agcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc
              ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcg
              ccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccgg
              atgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgagg
              cgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatcctta
              cccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagagaagg
              tcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatcaac
              gaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgcacc
              gttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaatt
              gaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcact
              ggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagg
              gatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaa
              agagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagc
              acgaggggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattc
              gttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtg
              ctgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacact
              gcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccg
              accattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcga
              gaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgc
              gttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctctt
              acggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccc
              tcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccgg
              atcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgag
              caacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgc
              gcagttaatacgcatggcatggatgaCCGATGGTAGTGTGGGGTCTCCCCATGCG
              AGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGT
              CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC
              TCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGC
              GAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAA
              CTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCC
              TTTTTGCGTGGCCAGTGCCAAGCTTGCATGCGTGC
Tet repressor taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct
SEQ ID        gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct
NO: 124       tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata
              atgcattctctagtgaaaaaccttgaggcataaaaaggctaattgattttcgagagtttcatactgatttctgtagg
              ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa
              aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg
              tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg
              ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagaca
              t
tetRitetA     cattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtga
promoters and actctagaaataattttgtttaactttaagaaggagatatacat
RBS and
leader region
SEQ ID NO
125:
trpE          atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttt
SEQ ID NO:    tcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaagatgatttaa
126           aaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttcc
              ggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaa
              actgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttt
              tgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcggcggcct
              gttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctgttt
              ttatctcgctgaaacgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctcc
              gaatgaagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgc
              cgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgta
              gtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctcgccgtttctctctgccct
              gcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggata
              atgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagattt
              acccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc
              atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgac
              ctggcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgat
              gcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga
              atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgac
              gcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctc
              ggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcg
              gaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggag
              acgttcta
TrpE          MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADIDSKD
SEQ ID NO:    DLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDNALPAGVENE
127           QSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRLLQNLLNVPKEEREA
              MFFGGLFSYDLVAGFENLPQLSAENSCPDFCFYLAETLMVIDHQKKST
              RIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVVSVPHMRCECN
              QSDEEFGGVVRLLQKAIRAGEIFQVVPSRRFSLPCPSPLAAYYVLKKS
              NPSPYMFFMQDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRA
              DGSLDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRY
              VADLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGTLSG
              APKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVIRSALVEN
              GIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF
trpD          atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtc
SEQ ID NO:    ataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcgacgatgagcaatccg
128           gtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcg
              tggcaagctgccaattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtca
              ggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaaca
              aacccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgccca
              ttttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccatt
              cttactacccagggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa
              cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttt
              tcagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatgaaaattcgcgg
              tgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccg
              gattatctgtttgccgatatcgtcggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcg
              tttgtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcg
              tcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagtta
              ggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaa
              aacccgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgttta
              tagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgca
              cagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcgaaattaag
              agctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggcaggcggaacaccgg
              aagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcgg
              cgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggta
              ctgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa
TrpD          MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQTLIERLATMS
SEQ ID NO:    NPVLMLSPGPGVPSEAGCMPELLTRLRGKLPIIGICLGHQAIVEAYGG
129           YVGQAGEILHGKASSIEHDGQAMFAGLTNPLPVARYHSLVGSNIPAG
              LTINAHFNGMVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLAW
              AQQKLEPTNTLQPILEKLYQAQTLSQQESHQLFSAVVRGELKPEQLAA
              ALVSMKIRGEHPNEIAGAATALLENAAPFPRPDYLFADIVGTGGDGSN
              SINISTASAFVAAACGLKVAKHGNRSVSSKSGSSDLLAAFGINLDMNA
              DKSRQALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLFNVLGPLI
              NPAHPPLALIGVYSPELVLPIAETLRVLGYQRAAVVHSGGMDEVSLH
              APTIVAELHDGEIKSYQLTAEDFGLTPYHQEQLAGGTPEENRDILTRLL
              QGKGDAAHEAAVAANVAMLMRLHGHEDLQANAQTVLEVLRSGSA
              YDRVTALAARG
trpC          atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaaccg
SEQ ID NO:    ctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggc
130           gtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgc
              cgccatttataaacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcct
              ccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg
              gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaatatcgccagcttgca
              gccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcgccatt
              gcattgggggcaaaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg
              cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggt
              gcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgatttgaacgccgc
              cgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttat
              gacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgca
              ggaagtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggaca
              aagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaatctgcgt
              gaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaactcttcccgcgcgcgattttca
              gcacatcgataaatatgtattcgacaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatg
              gtcaatcgcttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaaccg
              gctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctcggt
              tttccagacgctgcgcgcatattaa
TrpC          MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTRHFYDALQGA
SEQ ID NO:    RTAFILECKKASPSKGVIRDDFDPARIAAIYKHYASAISVLTDEKYFQG
131           SFDFLPIVSQIAPQPILCKDFIIDPYQIYLARYYQADACLLMLSVLDDEQ
              YRQLAAVAHSLEMGVLTEVSNEEELERAIALGAKVVGINNRDLRDLS
              IDLNRTRELAPKLGHNVTVISESGINTYAQVRELSHFANGFLIGSALM
              AHDDLNAAVRRVLLGENKVCGLTRGQDAKAAYDAGAIYGGLIFVAT
              SPRCVNVEQAQEVMAAAPLQYVGVFRNHDIADVADKAKVLSLAAV
              QLHGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDFQHIDKYVF
              DNGQGGSGQRFDWSLLNGQSLGNVLLAGGLGADNCVEAAQTGCAG
              LDFNSAVESQPGIKDARLLASVFQTLRAY
trpB          atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg
SEQ ID NO:    ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa
132           actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc
              tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga
              agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca
              gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc
              cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg
              aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc
              cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga
              aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc
              aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc
              accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa
              attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc
              actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa
              gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa
              aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa
              gcacgaggggaaatctga
TrpB          MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFND
SEQ ID NO:    LLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLG
133           QALLAKRMGKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDV
              ERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAH
              YMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGG
              GSNAIGMFADFINETDVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFG
              MKAPMMQTEDGQIEESYSISAGLDFPSVGPQHAYLNSTGRADYVSIT
              DDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQLLVVN
              LSGRGDKDIFTVHDILKARGEI
trpA          atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc
SEQ ID NO:    ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt
134           aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg
              ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc
              ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga
              ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg
              cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac
              ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg
              aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg
              cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc
              cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa
TrpA          MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALE
SEQ ID NO:    LGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGL
135           LMYANLVFNKGIDEFYAECEKVGVDSVLVADVPVEESAPFRQAALR
              HNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPL
              NHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKII
              EQHINEPEKMLAALKAFVQPMKAATRS

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 14 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 14 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 14 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 14 or a functional fragment thereof.

Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 123 through SEQ ID NO: 135. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 123 through SEQ ID NO: 135. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 123 through SEQ ID NO: 135.

Table 15 depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 15 also depicts an exemplary TnaA (tryptophanase from E. coli ) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodimetns, the sequence is deleted from the E coli chromosome to increase levels of tryptophan.

TABLE 15
Feedback resistant AroG and TrpE and tryptophanase sequences
Description          Sequence
Aro Gfbr: feedback   MNYQNDDLRIKEIKELLPPVALLEKFPATENAANTVAHARKAI
resistant 2-dehydro- HKILKGNDDRLLVVIGPCSIHDPVAAKEYATRLLTLREELQDE
3-                   LEIVMRVYFEKPRTTVGWKGLINDPHMDNSFQINDGLRIARK
deoxyphospho-        LLLDINDSGLPAAGEFLDMITLQYLADLMSWGAIGARTTESQ
heptonate aldolase   VHRELASGLSCPVGFKNGTDGTIKVAIDAINAAGAPHCFLSVT
from E. coli         KWGHSAIVNTSGNGDCHIILRGGKEPNYSAKHVAEVKEGLNK
SEQ ID NO: 136       AGLPAQVMIDFSHANSSKQFKKQMDVCTDVCQQIAGGEKAII
                     GVMVESHLVEGNQSLESGEPLAYGKSITDACIGWDDTDALLR
                     QLASAVKARRG
TrpEfbr: feedback    MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLEFADI
resistant            DSKDDLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDN
anthranilate         ALPAGVENEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRL
synthase             LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLSAENSCP
component I from     DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE
E. coli              LRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAI
SEQ ID NO: 137       RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN
                     DFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRD
                     LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA
                     DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT
                     LSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV
                     IRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRA
                     IATAHHAQETF
SerA: 2-             MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
oxoglutarate         DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
reductase from E.    NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
coli Nissle          ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE
SEQ ID NO: 137       SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
                     NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
                     HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
                     QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI
                     HENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA
                     DEDVAEKALQAMKAIPGTIRARLLY
SerAfbr: feedback    MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
resistant 2-         DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
oxoglutarate         NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
reductase from E.    ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE
coli Nissle          SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
SEQ ID NO: 139       NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
                     HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
                     QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI
                     AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA
                     DEDVAEKALQAMKAIPGTIRARLLY
TnaA:                MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE
tryptophanase from   DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE
E. coli              SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM
SEQ ID NO: 140       VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN
                     FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM
                     YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE
                     TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT
                     LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA
                     QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA
                     QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI
                     PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT
                     AKLKEV

In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. Accordingly, in one embodiment, one or more polypeptides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In another embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 136 through SEQ ID NO: 139.

In some embodiments, the endogenous TnaA polypeptide comprising SEQ ID NO: 140 is mutated or deleted.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Producing Kynurenic Acid

In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine--oxoglutarate transaminase.

In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the circulation. In some embodiments the TRP:KYNA ratio is increased. In some embodiments, TRP:KYNA ratio is decreased.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine--oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine--oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine aminotransferases.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenic acid from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37F and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with ID01. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TDO2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 (Kynurenine--oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 from homo sapiens). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine--oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cc1b1 and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of cc1b1 and/or cc1b2 and/or aadat and/or got2.

In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Producing Indole Tryptophan Metabolites and Tryptamine

In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of indole metabolites and/or tryptamine. Exemplary circuits for the production of indole metabolites/derivatives (e.g., FIG. 34, FIG. 35) are shown in FIG. 37A through FIG. 37H, FIG. 38A through FIG. 38F, and FIG. 39A though FIG. 39C, FIG. 43, and FIG. 44.

In some embodiments, the genetically engineered bacteria comprise genetic circuitry for converting tryptophan to tryptamine. In some embodiments, the engineered bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from Catharanthus roseus. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N- monooxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynurenine from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynureninic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g.,from homo sapiens or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tnaA (tryptophanase, e.g., from E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate (taken up through the diet). The genetically engineered bacteria comprise a gene sequence encoding pne2 (myrosinase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae), iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes), tynA (Monoamine oxidase, e.g., from E. coli ), iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana, cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana), nit1 (Nitrilase, e.g., from Arabidopsis thaliana), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. In some embodiments, the engineered bacteria produces tryptamine. Tryptophan is optionally produced from chorismate precursor, and the bacteria optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the bacteria comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.

In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-acetate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-propionate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises a circuit as described in FIG. 44, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH 1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-yl)pyruvate into indole-3-lactate).

In some embodiments, the engineered bacteria comprises genetic circuitry for the production of indole-3-propionic acid (IPA). In some embodiments, the engineered bacteria comprises gene sequence encoding tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or tryptamine synthesis(TrA) circuits. In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldH1/F1dH2: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; lad1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.

In some embodiments, the engineered bacteria comprise genetic circuitry for producing (indol-3-yl)pyruvate (IPyA). In some embodiments, the engineered bacteria comprise gene sequence encoing one or more of the following: tryptophan dehydrogenase (EC 1.4.1.19) (enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and ft)); Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) (converts (indol-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+); Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (F1dA) (converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) (convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA); Indole-3-lactate dehydratase (FldBC) (converts indole-3-lactate-CoA to indole-3-acrylyl-CoA); Indole-3-pyruvate decarboxylase (1pdC:) (converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d)); lad1: Indole-3-acetaldehyde dehydrogenase (coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA)); Tdc: Tryptophan decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In any of the described embodiments, any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA; thymidine dependence).

In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In in any of these embodiments the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, indole, indole acetic acid FICZ, indole-3-propionic acid, is under the control of an inducible promoter. Exemplary inducible promoters which may control the expression of the biosynthetic cassettes include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite characteristic of a disorder, such as liver damage or a metabolic disease, or that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Tryptamine

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole-3-acetic acid (IAA) via the enzymes tryptophan monooxygenase (IaaM) and indole-3- acetamide hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, see eg., FIG. 34, FIG. 35A and FIG. 35B.

A non-limiting example of such as strain is shown in FIG. 37A. Another non-limiting example of such as strain is shown in FIG. 39A. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Clostridium sporgenenes. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus.

Table 15, Table 16, and Table 17 lists exemplary sequences for tryptamine production in genetically engineered bacteria.

In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Indole-3-acetaldehyde and FICZ

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 37B.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and ipdC.

Further exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 37C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA.

In any of these embodiments, the genetically engineered bacteria which produce produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. Indole-3-acetic acid

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in Table 14. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole-3-aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter.

The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite , e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetic acid.

Non-limiting example of such gene sequence(s) are shown in FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E, and FIG. 39B and FIG. 39E.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1 (see, e.g., FIG. 38A).

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38B. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iadl and/or aao 1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iadl and/or aao 1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iad1 and/or aao1.

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and yuc2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and yuc2.

Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 38D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.

Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 38E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13 and nit1 and iaaH.

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38F. Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 39E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iad1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iad1.

In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole Acetic Acid and under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. Indole-3-acetonitrile

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3-acetonitrile from tryptophan is depicted in FIG. 16D.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13.

In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Indole-3-Propionic Acid (IPA)

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan. FIG. 43 and FIG. 44, and FIG. 39C depict schematics of exemplary circuits for the production of indole-3-propionic acid.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from Rubrivivax benzoatilyticus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase from Clostridum botulinum. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3-acrylate reductase. In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein.

The genetically engineered bacteria comprise a circuit, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole-3-lactate) (see, e.g., FIG. 44).

Another embodiment of the IPA producing strain is shown in FIG. 43.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul from Rhodobacter sphaeroides. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 (3-lactate dehydrogenase 1). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole-3-lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from Clostridium sporogenes). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH2.

In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites selected from tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acidand/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Indole

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37G and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA (tryptophanase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from E. coli.

In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-acetonitrile under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Other Indole Metabolites

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. Non-limiting example of such gene sequence(s) are shown FIG. 37H and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 from Arabidopsis thaliana.

In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing these metabolites under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Tryptophan Catabolic Pathway Enzymes

Table 16A and Table 16B comprise polypeptide and polynucleotide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure.

TABLE 16A
Tryptophan Pathway Catabolic Enzymes
Description          Sequence
TDC: Tryptophan      MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADYY
decarboxylase from   KNVETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQKDII
Catharanthus roseus  PGMTNWMSPNFYAFFPATVSSAAFLGEMLSTALNSVGFTWV
SEQ ID NO: 141       SSPAATELEMIVMDWLAQILKLPKSFMFSGTGGGVIQNTTSES
                     ILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKTCKLA
                     GIYPNNIRLIPTTVETDFGISPQVLRKMVEDDVAAGYVPLFLC
                     ATLGTTSTTATDPVDSLSEIANEFGIWIHVDAAYAGSACICPEF
                     RHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQPHLLLRAL
                     TTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRS
                     YGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEIVVPRNFSLVC
                     FRLKPDVSSLHVEEVNKKLLDMLNSTGRVYMTHTIVGGIYML
                     RLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA
TDC: Tryptophan      MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDT
decarboxylase from   VFYDDHSFVKHSPYLRTFIQNPNHIGCHTYDKADILFGGTFDIE
Clostridium          RELIQLLAIDVLNGNDEEFDGYVTQGGTEANIQAMWVYRNY
sporogenes           FKKERKAKHEEIAIITSADTHYSAYKGSDLLNIDIIKVPVDFYS
SEQ ID NO: 142       RKIQENTLDSIVKEAKEIGKKYFIVISNMGTTMFGSVDDPDLY
                     ANIFDKYNLEYKIHVDGAFGGFIYPIDNKECKTDFSNKNVSSIT
                     LDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVTLSG
                     SRSGSNAVAIWMVLASYGPYGWMEKINKLRNRTKWLCKQL
                     NDMRIKYYKEDSMNIVTIEEQYVNKEIAEKYFLVPEVHNPTN
                     NWYKIVVMEHVELDILNSLVYDLRKFNKEHLKAM
TYNA: Monoamine      MGSPSLYSARKTTLALAVALSFAWQAPVFAHGGEAHMVPM
oxidase from E. coli DKTLKEFGADVQWDDYAQLFTLIKDGAYVKVKPGAQTAIVN
SEQ ID NO: 143       GQPLALQVPVVMKDNKAWVSDTFINDVFQSGLDQTFQVEKR
                     PHPLNALTADEIKQAVEIVKASADFKPNTRFTEISLLPPDKEAV
                     WAFALENKPVDQPRKADVIMLDGKHIIEAVVDLQNNKLLSW
                     QPIKDAHGMVLLDDFASVQNIINNSEEFAAAVKKRGITDAKK
                     VITTPLTVGYFDGKDGLKQDARLLKVISYLDVGDGNYWAHPI
                     ENLVAVVDLEQKKIVKIEEGPVVPVPMTARPFDGRDRVAPAV
                     KPMQIIEPEGKNYTITGDMIHWRNWDFHLSMNSRVGPMISTV
                     TYNDNGTKRKVMYEGSLGGMIVPYGDPDIGWYFKAYLDSGD
                     YGMGTLTSPIARGKDAPSNAVLLNETIADYTGVPMEIPRAIAV
                     FERYAGPEYKHQEMGQPNVSTERRELVVRWISTVGNYDYIFD
                     WIFHENGTIGIDAGATGIEAVKGVKAKTMHDETAKDDTRYGT
                     LIDHNIVGTTHQHIYNFRLDLDVDGENNSLVAMDPVVKPNTA
                     GGPRTSTMQVNQYNIGNEQDAAQKFDPGTIRLLSNPNKENRM
                     GNPVSYQIIPYAGGTHPVAKGAQFAPDEWIYHRLSFMDKQLW
                     VTRYHPGERFPEGKYPNRSTHDTGLGQYSKDNESLDNTDAV
                     VWMTTGTTHVARAEEWPIMPTEWVHTLLKPWNFFDETPTLG
                     ALKKDK
AAO1: Indole-3-      MGEKAIDEDKVEAMKSSKTSLVFAINGQRFELELSSIDPSTTL
acetaldehyde oxidase VDFLRNKTPFKSVKLGCGEGGCGACVVLLSKYDPLLEKVDEF
from Arabidopsis     TISSCLTLLCSIDGCSITTSDGLGNSRVGFHAVHERIAGFHATQ
thaliana             CGFCTPGMSVSMFSALLNADKSHPPPRSGFSNLTAVEAEKAV
SEQ ID NO: 144       SGNLCRCTGYRPLVDACKSFAADVDIEDLGFNAFCKKGENRD
                     EVLRRLPCYDHTSSHVCTFPEFLKKEIKNDMSLHSRKYRWSSP
                     VSVSELQGLLEVENGLSVKLVAGNTSTGYYKEEKERKYERFI
                     DIRKIPEFTMVRSDEKGVELGACVTISKAIEVLREEKNVSVLA
                     KIATHMEKIANRFVRNTGTIGGNIMMAQRKQFPSDLATILVA
                     AQATVKIMTSSSSQEQFTLEEFLQQPPLDAKSLLLSLEIPSWHS
                     AKKNGSSEDSILLFETYRAAPRPLGNALAFLNAAFSAEVTEAL
                     DGIVVNDCQLVFGAYGTKHAHRAKKVEEFLTGKVISDEVLM
                     EAISLLKDEIVPDKGTSNPGYRSSLAVTFLFEFFGSLTKKNAKT
                     TNGWLNGGCKEIGFDQNVESLKPEAMLSSAQQIVENQEHSPV
                     GKGITKAGACLQASGEAVYVDDIPAPENCLYGAFIYSTMPLA
                     RIKGIRFKQNRVPEGVLGIITYKDIPKGGQNIGTNGFFTSDLLF
                     AEEVTHCAGQIIAFLVADSQKHADIAANLVVIDYDTKDLKPPI
                     LSLEEAVENFSLFEVPPPLRGYPVGDITKGMDEAEHKILGSMS
                     FGSQYFFYMETQTALAVPDEDNCMVVYSSTQTPEFVHQTIAG
                     CLGVPENNVRVITRRVGGGFGGKAVKSMPVAAACALAASK
                     MQRPVRTYVNRKTDMITTGGRHPMKVTYSVGFKSNGKITAL
                     DVEVLLDAGLTEDISPLMPKGIQGALMKYDWGALSFNVKVC
                     KTNTVSRTALRAPGDVQGSYIGEAIIEKVASYLSVDVDEIRKV
                     NLHTYESLRLFHSAKAGEFSEYTLPLLWDRIDEFSGFNKRRKV
                     VEEFNASNKWRKRGISRVPAVYAVNMRSTPGRVSVLGDGSIV
                     VEVQGIEIGQGLWTKVKQMAAYSLGLIQCGTTSDELLKKIRVI
                     QSDTLSMVQGSMTAGSTTSEASSEAVRICCDGLVERLLPVKT
                     ALVEQTGGPVTWDSLISQAYQQSINMSVSSKYMPDSTGEYLN
                     YGIAASEVEVNVLTGETTILRTDIIYDCGKSLNPAVDLGQIEGA
                     FVQGLGFFMLEEFLMNSDGLVVTDSTWTYKIPTVDTIPRQFN
                     VEILNSGQHKNRVLSSKASGEPPLLLAASVHCAVRAAVKEAR
                     KQILSWNSNKQGTDMYFELPVPATMPIVKEFCGLDVVEKYLE
                     WKIQQRKNV
ARO9: L-tryptophan   MTAGSAPPVDYTSLKKNFQPFLSRRVENRSLKSFWDASDISD
amino transferase    DVIELAGGMPNERFFPIESMDLKISKVPFNDNPKWHNSFTTAH
from S. cerevisae    LDLGSPSELPIARSFQYAETKGLPPLLHFVKDFVSRINRPAFSD
SEQ ID NO: 145       ETESNWDVILSGGSNDSMFKVFETICDESTTVMIEEFTFTPAM
                     SNVEATGAKVIPIKMNLTFDRESQGIDVEYLTQLLDNWSTGP
                     YKDLNKPRVLYTIATGQNPTGMSVPQWKREKIYQLAQRHDF
                     LIVEDDPYGYLYFPSYNPQEPLENPYHSSDLTTERYLNDFLMK
                     SFLTLDTDARVIRLETFSKIFAPGLRLSFIVANKFLLQKILDLAD
                     ITTRAPSGTSQAIVYSTIKAMAESNLSSSLSMKEAMFEGWIRW
                     IMQIASKYNHRKNLTLKALYETESYQAGQFTVMEPSAGMFIII
                     KINWGNFDRPDDLPQQMDILDKFLLKNGVKVVLGYKMAVCP
                     NYSKQNSDFLRLTIAYARDDDQLIEASKRIGSGIKEFFDNYKS
aspC: aspartate      MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKT
amino transferase    PVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKG
from E. coli         SALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSN
SEQ ID NO: 146       PSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEA
                     QAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLF
                     DFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYN
                     ERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASV
                     VATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGAN
                     RDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVA
                     GMTPDNMAPLCEAIVAVL
TAA1: L-tryptophan-  MVKLENSRKPEKISNKNIPMSDFVVNLDHGDPTAYEEYWRK
pyruvate             MGDRCTVTIRGCDLMSYFSDMTNLCWFLEPELEDAIKDLHGV
amino transferase    VGNAATEDRYIVVGTGSTQLCQAAVHALSSLARSQPVSVVA
from Arabidopsis     AAPFYSTYVEETTYVRSGMYKWEGDAWGFDKKGPYIELVTS
thaliana             PNNPDGTIRETVVNRPDDDEAKVIHDFAYYWPHYTPITRRQD
SEQ ID NO: 147       HDIMLFTFSKITGHAGSRIGWALVKDKEVAKKMVEYIIVNSIG
                     VSKESQVRTAKILNVLKETCKSESESENFFKYGREMMKNRWE
                     KLREVVKESDAFTLPKYPEAFCNYFGKSLESYPAFAWLGTKE
                     ETDLVSELRRHKVMSRAGERCGSDKKHVRVSMLSREDVFNV
                     FLERLANMKLIKSIDL
STAO: L-tryptophan   MTAPLQDSDGPDDAIGGPKQVTVIGAGIAGLVTAYELERLGH
oxidase from         HVQIIEGSDDIGGRIHTHRFSGAGGPGPFAEMGAMRIPAGHRL
streptomyces sp.     TMHYIAELGLQNQVREFRTLFSDDAAYLPSSAGYLRVREAHD
TP-A0274             TLVDEFATGLPSAHYRQDTLLFGAWLDASIRAIAPRQFYDGL
SEQ ID NO: 148       HNDIGVELLNLVDDIDLTPYRCGTARNRIDLHALFADHPRVR
                     ASCPPRLERFLDDVLDETSSSIVRLKDGMDELPRRLASRIRGKI
                     SLGQEVTGIDVHDDTVTLTVRQGLRTVTRTCDYVVCTIPFTVL
                     RTLRLTGFDQDKLDIVHETKYWPATKIAFHCREPFWEKDGIS
                     GGASFTGGHVRQTYYPPAEGDPALGAVLLASYTIGPDAEALA
                     RMDEAERDALVAKELSVMHPELRRPGMVLAVAGRDWGARR
                     WSRGAATVRWGQEAALREAERRECARPQKGLFFAGEHCSSK
                     PAWIEGAIESAIDAAHEIEWYEPRASRVFAASRLSRSDRSA
ipdC: Indole-3-      MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDS
pyruvate             PDICWVGCANELNASYAADGYARCKGFAALLTTFGVGELSA
decarboxylase from   MNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLGDGEFR
Enterobacter cloacae HFYHMSEPITVAQAVLTEQNACYEIDRVLTTMLRERRPGYLM
SEQ ID NO: 149       LPADVAKKAATPPVNALTHKQAHADSACLKAFRDAAENKLA
                     MSKRTALLADFLVLRHGLKHALQKWVKEVPMAHATMLMG
                     KGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVLCVGTRFT
                     DTLTAGFTHQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIET
                     LVELCKQHVHAGLMSSSSGAIPFPQPDGSLTQENFWRTLQTFI
                     RPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGSIGYTLAA
                     AFGAQTACPNRRVIVLTGDGAAQLTIQELGSMLRDKQHPIILV
                     LNNEGYTVERAIHGAEQRYNDIALWNWTHIPQALSLDPQSEC
                     WRVSEAEQLADVLEKVAHHERLSLIEVMLPKADIPPLLGALT
                     KALEACNNA
IAD1: Indole-3-      MPTLNLDLPNGIKSTIQADLFINNKFVPALDGKTFATINPSTGK
acetaldehyde         EIGQVAEASAKDVDLAVKAAREAFETTWGENTPGDARGRLLI
dehydrogenase from   KLAELVEANIDELAAIESLDNGKAFSIAKSFDVAAVAANLRY
Ustilago maydis      YGGWADKNHGKVMEVDTKRLNYTRHEPIGVCGQIIPWNFPL
SEQ ID NO: 150       LMFAWKLGPALATGNTIVLKTAEQTPLSAIKMCELIVEAGFPP
                     GVVNVISGFGPVAGAAISQHMDIDKIAFTGSTLVGRNIMKAA
                     ASTNLKKVTLELGGKSPNIIFKDADLDQAVRWSAFGIMFNHG
                     QCCCAGSRVYVEESIYDAFMEKMTAHCKALQVGDPFSANTF
                     QGPQVSQLQYDRIMEYIESGKKDANLALGGVRKGNEGYFIEP
                     TIFTDVPHDAKIAKEEIFGPVVVVSKFKDEKDLIRIANDSIYGL
                     AAAVFSRDISRAIETAHKLKAGTVWVNCYNQLIPQVPFGGYK
                     ASGIGRELGEYALSNYTNIKAVHVNLSQPAPI
YUC2: indole-3-      MEFVTETLGKRIHDPYVEETRCLMIPGPIIVGSGPSGLATAACL
pyruvate             KSRDIPSLILERSTCIASLWQHKTYDRLRLHLPKDFCELPLMPF
monoxygenase from    PSSYPTYPTKQQFVQYLESYAEHFDLKPVFNQTVEEAKFDRR
Arabidopsis thaliana CGLWRVRTTGGKKDETMEYVSRWLVVATGENAEEVMPEID
SEQ ID NO: 151       GIPDFGGPILHTSSYKSGEIFSEKKILVVGCGNSGMEVCLDLCN
                     FNALPSLVVRDSVHVLPQEMLGISTFGISTSLLKWFPVHVVDR
                     FLLRMSRLVLGDTDRLGLVRPKLGPLERKIKCGKTPVLDVGT
                     LAKIRSGHIKVYPELKRVMHYSAEFVDGRVDNFDAIILATGY
                     KSNVPMWLKGVNMFSEKDGFPHKPFPNGWKGESGLYAVGF
                     TKLGLLGAAIDAKKIAEDIEVQRHFLPLARPQHC
IaaM: Tryptophan 2-  MYDHFNSPSIDILYDYGPFLKKCEMTGGIGSYSAGTPTPRVAI
monooxygenase from   VGAGISGLVAATELLRAGVKDVVLYESRDRIGGRVWSQVFD
Pseudomonas          QTRPRYIAEMGAMRFPPSATGLFHYLKKFGISTSTTFPDPGVV
savastanoi           DTELHYRGKRYHWPAGKKPPELFRRVYEGWQSLLSEGYLLE
SEQ ID NO: 152       GGSLVAPLDITAMLKSGRLEEAAIAWQGWLNVFRDCSFYNAI
                     VCIFTGRHPPGGDRWARPEDFELFGSLGIGSGGFLPVFQAGFT
                     EILRMVINGYQSDQRLIPDGISSLAARLADQSFDGKALRDRVC
                     FSRVGRISREAEKIIIQTEAGEQRVFDRVIVTSSNRAMQMIHCL
                     TDSESFLSRDVARAVRETHLTGSSKLFILTRTKFWIKNKLPTTI
                     QSDGLVRGVYCLDYQPDEPEGHGVVLLSYTWEDDAQKMLA
                     MPDKKTRCQVLVDDLAAIHPTFASYLLPVDGDYERYVLHHD
                     WLTDPHSAGAFKLNYPGEDVYSQRLFFQPMTANSPNKDTGL
                     YLAGCSCSFAGGWIEGAVQTALNSACAVLRSTGGQLSKGNPL
                     DCINASYRY
iaaH:                MHEIITLESLCQALADGEIAAAELRERALDTEARLARLNCFIRE
Indoleacetamide      GDAVSQFGEADHAMKGTPLWGMPVSFKDNICVRGLPLTAGT
hydrolase from       RGMSGFVSDQDAAIVSQLRALGAVVAGKNNMHELSFGVTSI
Pseudomonas          NPHWGTVGNPVAPGYCAGGSSGGSAAAVASGIVPLSVGTDT
savastanoi           GGSIRIPAAFCGITGFRPTTGRWSTAGIIPVSHTKDCVGLLTRT
SEQ ID NO: 153       AGDAGFLYGLLSGKQQSFPLSRTAPCRIGLPVSMWSDLDGEV
                     ERACVNALSLLRKTGFEFIEIDDADIVELNQTLTFTVPLYEFFA
                     DLAQSLLSLGWKHGIHHIFAQVDDANVKGIINHHLGEGAIKP
                     AHYLSSLQNGELLKRKMDELFARHNIELLGYPTVPCRVPHLD
                     HADRPEFFSQAIRNTDLASNAMLPSITIPVGPEGRLPVGLSFDA
                     LRGRDALLLSRVSAIEQVLGFVRKVLPHTT
TrpDH: Tryptophan    MLLFETVREMGHEQVLFCHSKNPEIKAIIAIHDTTLGPAMGAT
dehydrogenase from   RILPYINEEAALKDALRLSRGMTYKAACANIPAGGGKAVIIAN
Nostoc punctiforme   PENKTDDLLRAYGRFVDSLNGRFITGQDVNITPDDVRTISQET
NIES-2108            KYVVGVSEKSGGPAPITSLGVFLGIKAAVESRWQSKRLDGMK
SEQ ID NO: 154       VAVQGLGNVGKNLCRHLHEHDVQLFVSDVDPIKAEEVKRLF
                     GATVVEPTEIYSLDVDIFAPCALGGILNSHTIPFLQASIIAGAAN
                     NQLENEQLHSQMLAKKGILYSPDYVINAGGLINVYNEMIGYD
                     EEKAFKQVHNIYDTLLAIFEIAKEQGVTTNDAARRLAEDRINN
                     SKRSKSKAIAA
CYP79B2:             MNTFTSNSSDLTTTATETSSFSTLYLLSTLQAFVAITLVMLLKK
tryptophan N-        LMTDPNKKKPYLPPGPTGWPIIGMIPTMLKSRPVFRWLHSIMK
monooxygenase from   QLNTEIACVKLGNTHVITVTCPKIAREILKQQDALFASRPLTY
Arabidopsis thaliana AQKILSNGYKTCVITPFGDQFKKMRKVVMTELVCPARHRWL
SEQ ID NO: 155       HQKRSEENDHLTAWVYNMVKNSGSVDFRFMTRHYCGNAIK
                     KLMFGTRTFSKNTAPDGGPTVEDVEHMEAMFEALGFTFAFCI
                     SDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR
                     EGKRTQIEDFLDIFISIKDEQGNPLLTADEIKPTIKELVMAAPDN
                     PSNAVEWAMAEMVNKPEILRKAMEEIDRVVGKERLVQESDIP
                     KLNYVKAILREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGS
                     QVLLSRYGLGRNPKVWADPLCFKPERHLNECSEVTLTENDLR
                     FISFSTGKRGCAAPALGTALTTMMLARLLQGFTWKLPENETR
                     VELMESSHDMFLAKPLVMVGDLRLPEHLYPTVK
CYP79B3:             MDTLASNSSDLTTKSSLGMSSFTNMYLLTTLQALAALCFLMI
tryptophan N-        LNKIKSSSRNKKLHPLPPGPTGFPIVGMIPAMLKNRPVFRWLH
monooxygenase from   SLMKELNTEIACVRLGNTHVIPVTCPKIAREIFKQQDALFASRP
Arabidopsis thaliana LTYAQKILSNGYKTCVITPFGEQFKKMRKVIMTEIVCPARHR
SEQ ID NO: 156       WLHDNRAEETDHLTAWLYNMVKNSEPVDLRFVTRHYCGNA
                     IKRLMFGTRTFSEKTEADGGPTLEDIEHMDAMFEGLGFTFAFC
                     ISDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR
                     EGKRTQIEDFLDIFISIKDEAGQPLLTADEIKPTIKELVMAAPDN
                     PSNAVEWAIAEMINKPEILHKAMEEIDRVVGKERFVQESDIPK
                     LNYVKAIIREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGSQV
                     LLSRYGLGRNPKVWSDPLSFKPERHLNECSEVTLTENDLRFIS
                     FSTGKRGCAAPALGTAITTMMLARLLQGFKWKLAGSETRVE
                     LMESSHDMFLSKPLVLVGELRLSEDLYPMVK
CYP71A13 :           MSNIQEMEMILSISLCLTTLITLLLLRRFLKRTATKVNLPPSPW
indoleacetaldoxime   RLPVIGNLHQLSLHPHRSLRSLSLRYGPLMLLHFGRVPILVVSS
dehydratase from     GEAAQEVLKTHDHKFANRPRSKAVHGLMNGGRDVVFAPYG
Arabidopis thaliana  EYWRQMKSVCILNLLTNKMVESFEKVREDEVNAMIEKLEKA
SEQ ID NO: 157       SSSSSSENLSELFITLPSDVTSRVALGRKHSEDETARDLKKRVR
                     QIMELLGEFPIGEYVPILAWIDGIRGFNNKIKEVSRGFSDLMDK
                     VVQEHLEASNDKADFVDILLSIEKDKNSGFQVQRNDIKFMILD
                     MFIGGTSTTSTLLEWTMTELIRSPKSMKKLQDEIRSTIRPHGSY
                     IKEKEVENMKYLKAVIKEVLRLHPSLPMILPRLLSEDVKVKGY
                     NIAAGTEVIINAWAIQRDTAIWGPDAEEFKPERHLDSGLDYHG
                     KNLNYIPFGSGRRICPGINLALGLAEVTVANLVGRFDWRVEA
                     GPNGDQPDLTEAIGIDVCRKFPLIAFPSSVV
PEN2: myrosinase     MAHLQRTFPTEMSKGRASFPKGFLFGTASSSYQYEGAVNEGA
from Arabidopsis     RGQSVWDHFSNRFPHRISDSSDGNVAVDFYHRYKEDIKRMK
thaliana             DINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLA
SEQ ID NO: 158       NEITPLVTIFHWDIPQDLEDEYGGFLSEQIIDDFRDYASLCFERF
                     GDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYVNGASV
                     AGMSGYEAYIVSHNMLLAHAEAVEVFRKCDHIKNGQIGIAHN
                     PLWYEPYDPSDPDDVEGCNRAMDFMLGWHQHPTACGDYPE
                     TMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKH
                     VDPTQPTWRTDQGVDWMKTNIDGKQIAKQGGSEWSFTYPTG
                     LRNILKYVKKTYGNPPILITENGYGEVAEQSQSLYMYNPSIDT
                     ERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSLLDNFEWNSGY
                     GVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTS
                     KKEEKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVV
                     SATVGTSLFFKGANN
Nit1: Nitrilase from MSSTKDMSTVQNATPFNGVAPSTTVRVTIVQSSTVYNDTPATI
Arabidopsis thaliana DKAEKYIVEAASKGAELVLFPEGFIGGYPRGFRFGLAVGVHN
SEQ ID NO: 159       EEGRDEFRKYHASAIHVPGPEVARLADVARKNHVYLVMGAI
                     EKEGYTLYCTVLFFSPQGQFLGKHRKLMPTSLERCIWGQGDG
                     STIPVYDTPIGKLGAAICWENRMPLYRTALYAKGIELYCAPTA
                     DGSKEWQSSMLHIAIEGGCFVLSACQFCQRKHFPDHPDYLFT
                     DWYDDKEHDSIVSQGGSVIISPLGQVLAGPNFESEGLVTADID
                     LGDIARAKLYFDSVGHYSRPDVLHLTVNEHPRKSVTFVTKVE
                     KAEDDSNK
IDO1: indoleamine    MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFI
2,3-dioxygenase from AKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLG
homo sapiens         CITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVY
SEQ ID NO: 160       ADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVS
                     LLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKA
                     LQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGF
                     WEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQ
                     DMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDA
                     CVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAK
                     GTGGTDLMNFLKTVRSTTEKSLLKEG
TDO2: tryptophan     MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLIY
2,3-dioxygenase from GNYLHLEKVLNAQELQSETKGNKIHDEHLFIITHQAYELWFK
homo sapiens         QILWELDSVREIFQNGHVRDERNMLKVVSRMHRVSVILKLLV
SEQ ID NO: 161       QQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIGVLQ
                     NMRVPYNRRHYRDNFKGEENELLLKSEQEKTLLELVEAWLE
                     RTPGLEPHGFNFWGKLEKNITRGLEEEFIRIQAKEESEEKEEQV
                     AEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRALQGALMIY
                     FYREEPRFQVPFQLLTSLMDIDSLMTKWRYNHVCMVHRMLG
                     SKAGTGGSSGYHYLRSTVSDRYKVFVDLFNLSTYLIPRHWIPK
                     MNPTIHKFLYTAEYCDSSYFSSDESD
BNA2: indoleamine    MNNTSITGPQVLHRTKMRPLPVLEKYCISPHHGFLDDRLPLTR
2,3-dioxygenase from LSSKKYMKWEEIVADLPSLLQEDNKVRSVIDGLDVLDLDETIL
S. cerevisiae        GDVRELRRAYSILGFMAHAYIWASGTPRDVLPECIARPLLETA
SEQ ID NO: 162       HILGVPPLATYSSLVLWNFKVTDECKKTETGCLDLENITTINTF
                     TGTVDESWFYLVSVRFEKIGSACLNHGLQILRAIRSGDKGDA
                     NVIDGLEGLAATIERLSKALMEMELKCEPNVFYFKIRPFLAGW
                     TNMSHMGLPQGVRYGAEGQYRIFSGGSNAQSSLIQTLDILLG
                     VKHTANAAHSSQGDSKINYLDEMKKYMPREHREFLYHLESV
                     CNIREYVSRNASNRALQEAYGRCISMLKIFRDNHIQIVTKYIIL
                     PSNSKQHGSNKPNVLSPIEPNTKASGCLGHKVASSKTIGTGGT
                     RLMPFLKQCRDETVATADIKNEDKN
Afmid: Kynurenine    MAFPSLSAGQNPWRNLSSEELEKQYSPSRWVIHTKPEEVVGN
formamidase from     FVQIGSQATQKARATRRNQLDVPYGDGEGEKLDIYFPDEDSK
mouse                AFPLFLFLHGGYWQSGSKDDSAFMVNPLTAQGIVVVIVAYDI
SEQ ID NO: 163       APKGTLDQMVDQVTRSVVFLQRRYPSNEGIYLCGHSAGAHL
                     AAMVLLARWTKHGVTPNLQGFLLVSGIYDLEPLIATSQNDPL
                     RMTLEDAQRNSPQRHLDVVPAQPVAPACPVLVLVGQHDSPE
                     FHRQSKEFYETLLRVGWKASFQQLRGVDHFDIIENLTREDDV
                     LTQIILKTVFQKL
BNA3: kynurenine--   MKQRFIRQFTNLMSTSRPKVVANKYFTSNTAKDVWSLTNEA
oxoglutarate         AAKAANNSKNQGRELINLGQGFFSYSPPQFAIKEAQKALDIPM
transaminase from S. VNQYSPTRGRPSLINSLIKLYSPIYNTELKAENVTVTTGANEGI
cerevisae            LSCLMGLLNAGDEVIVFEPFFDQYIPNIELCGGKVVYVPINPPK
SEQ ID NO: 164       ELDQRNTRGEEWTIDFEQFEKAITSKTKAVIINTPHNPIGKVFT
                     REELTTLGNICVKHNVVIISDEVYEHLYFTDSFTRIATLSPEIGQ
                     LTLTVGSAGKSFAATGWRIGWVLSLNAELLSYAAKAHTRICF
                     ASPSPLQEACANSINDALKIGYFEKMRQEYINKFKIFTSIFDEL
                     GLPYTAPEGTYFVLVDFSKVKIPEDYPYPEEILNKGKDFRISH
                     WLINELGVVAIPPTEFYIKEHEKAAENLLRFAVCKDDAYLEN
                     AVERLKLLKDYL
GOT2: Aspartate      MALLHSGRVLPGIAAAFHPGLAAAASARASSWWTHVEMGPP
aminotransferase,    DPILGVTEAFKRDTNSKKMNLGVGAYRDDNGKPYVLPSVRK
mitochondrial from   AEAQIAAKNLDKEYLPIGGLAEFCKASAELALGENSEVLKSG
homo sapiens         RFVTVQTISGTGALRIGASFLQRFFKFSRDVFLPKPTWGNHTPI
SEQ ID NO: 165       FRDAGMQLQGYRYYDPKTCGFDFTGAVEDISKIPEQSVLLLH
                     ACAHNPTGVDPRPEQWKEIATVVKKRNLFAFFDMAYQGFAS
                     GDGDKDAWAVRHFIEQGINVCLCQSYAKNMGLYGERVGAFT
                     MVCKDADEAKRVESQLKILIRPMYSNPPLNGARIAAAILNTPD
                     LRKQWLQEVKVMADRIIGMRTQLVSNLKKEGSTHNWQHITD
                     QIGMFCFTGLKPEQVERLIKEFSIYMTKDGRISVAGVTSSNVG
                     YLAHAIHQVTK
AADAT:               MNYARFITAASAARNPSPIRTMTDILSRGPKSMISLAGGLPNP
Kynurenine/alpha-    NMFPFKTAVITVENGKTIQFGEEMMKRALQYSPSAGIPELLSW
aminoadipate         LKQLQIKLHNPPTIHYPPSQGQMDLCVTSGSQQGLCKVFEMII
aminotransferase,    NPGDNVLLDEPAYSGTLQSLHPLGCNIINVASDESGIVPDSLR
mitochondrial        DILSRWKPEDAKNPQKNTPKFLYTVPNGNNPTGNSLTSERKK
SEQ ID NO: 166       EIYELARKYDFLIIEDDPYYFLQFNKFRVPTFLSMDVDGRVIRA
                     DSFSKITSSGLRIGFLTGPKPLIERVILHIQVSTLHPSTFNQLMIS
                     QLLHEWGEEGFMAHVDRVIDFYSNQKDAILAAADKWLTGLA
                     EWHVPAAGMFLWIKVKGINDVKELIEEKAVKMGVLMLPGN
                     AFYVDSSAPSPYLRASFSSASPEQMDVAFQVLAQLIKESL
CCLB1: Kynurenine-   MAKQLQARRLDGIDYNPWVEFVKLASEHDVVNLGQGFPDFP
-oxoglutarate        PPDFAVEAFQHAVSGDFMLNQYTKTFGYPPLTKILASFFGELL
transaminase 1 from  GQEIDPLRNVLVTVGGYGALFTAFQALVDEGDEVIIIEPFFDC
homo sapiens         YEPMTMMAGGRPVFVSLKPGPIQNGELGSSSNWQLDPMELA
SEQ ID NO: 167       GKFTSRTKALVLNTPNNPLGKVFSREELELVASLCQQHDVVCI
                     TDEVYQWMVYDGHQHISIASLPGMWERTLTIGSAGKTFSATG
                     WKVGWVLGPDHIMKHLRTVHQNSVFHCPTQSQAAVAESFER
                     EQLLFRQPSSYFVQFPQAMQRCRDHMIRSLQSVGLKPIIPQGS
                     YFLITDISDFKRKMPDLPGAVDEPYDRRFVKWMIKNKGLVAI
                     PVSIFYSVPHQKHFDHYIRFCFVKDEATLQAMDEKLRKWKVE
                     L
CCLB2: kynurenine--  MFLAQRSLCSLSGRAKFLKTISSSKILGFSTSAKMSLKFTNAKR
oxoglutarate         IEGLDSNVWIEFTKLAADPSVVNLGQGFPDISPPTYVKEELSKI
transaminase 3 from  AAIDSLNQYTRGFGHPSLVKALSYLYEKLYQKQIDSNKEILVT
homo sapiens         VGAYGSLFNTIQALIDEGDEVILIVPFYDCYEPMVRMAGATPV
SEQ ID NO: 168       FIPLRSKPVYGKRWSSSDWTLDPQELESKFNSKTKAIILNTPHN
                     PLGKVYNREELQVIADLCIKYDTLCISDEVYEWLVYSGNKHL
                     KIATFPGMWERTITIGSAGKTFSVTGWKLGWSIGPNHLIKHLQ
                     TVQQNTIYTCATPLQEALAQAFWIDIKRMDDPECYFNSLPKEL
                     EVKRDRMVRLLESVGLKPIVPDGGYFIIADVSLLDPDLSDMK
                     NNEPYDYKFVKWMTKHKKLSAIPVSAFCNSETKSQFEKFVRF
                     CFIKKDSTLDAAEEIIKAWSVQKS
TnaA: tryptophanase  MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE
from E. coli         DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE
SEQ ID NO: 140       SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM
                     VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN
                     FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAM
                     YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE
                     TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT
                     LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA
                     QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFP
                     AQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRL
                     TIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRH
                     FTAKLKEV
Trp                  MTATTISIETVPQAPAAGTKTNGTSGKYNPRTYLSDRAKVTEI
aminotransferase     DGSDAGRPNPDTFPFNSITLNLKPPLGLPESSNNMPVSITIEDPD
(EC 2.6.1.27);       LATALQYAPSAGIPKLREWLADLQAHVHERPRGDYAISVGSG
tryptophan           SQDLMFKGFQAVLNPGDPVLLETPMYSGVLPALRILKADYAE
aminotransferase     VDVDDQGLSAKNLEKVLSEWPADKKRPRVLYTSPIGSNPSGC
[Cryptococcus        SASKERKLEVLKVCKKYDVLIFEDDPYYYLAQELIPSYFALEK
deuterogattii R265]  QVYPEGGHVVRFDSFSKLLSAGMRLGFATGPKEILHAIDVSTA
SEQ ID NO: 169       GANLHTSAVSQGVALRLMQYWGIEGFLAHGRAVAKLYTERR
                     AQFEATAHKYLDGLATWVSPVAGMFLWIDLRPAGIEDSYELI
                     RHEALAKGVLGVPGMAFYPTGRKSSHVRVSFSIVDLEDESDL
                     GFQRLAEAIKDKRKALGLA
Tryptophan           MSQVIKKKRNTFMIGTEYILNSTQLEEAIKSFVHDFCAEKHEIH
Decarboxylase (EC    DQPVVVEAKEHQEDKIKQIKIPEKGRPVNEVVSEMMNEVYRY
4.1.1.28) Chain A,   RGDANHPRFFSFVPGPASSVSWLGDIMTSAYNIHAGGSKLAP
Ruminococcus         MVNCIEQEVLKWLAKQVGFTENPGGVFVSGGSMANITALTA
Gnavus               ARDNKLTDINLHLGTAYISDQTHSSVAKGLRIIGITDSRIRRIPT
SEQ ID NO: 170       NSHFQMDTTKLEEAIETDKKSGYIPFVVIGTAGTTNTGSIDPLT
                     EISALCKKHDMWFHIDGAYGASVLLSPKYKSLLTGTGLADSIS
                     WDAHKWLFQTYGCAMVLVKDIRNLFHSFHVNPEYLKDLEN
                     DIDNVNTWDIGMELTRPARGLKLWLTLQVLGSDLIGSAIEHG
                     FQLAVWAEEALNPKKDWEIVSPAQMAMINFRYAPKDLTKEE
                     QDILNEKISHRILESGYAAIFTTVLNGKTVLRICAIHPEATQED
                     MQHTIDLLDQYGREIYTEMKKa

TABLE 16B
Tryptophan Pathway Catabolic Enzymes
Description           Sequence
Trp                   ATGACGGCAACTACAATTTCTATTGAGACCGTACCTC
aminotransferase      AGGCCCCGGCGGCGGGGACCAAAACTAATGGGACTT
(EC 2.6.1.27);        CAGGAAAATACAACCCCCGCACTTACCTGTCCGACC
tryptophan            GCGCCAAAGTCACTGAGATTGATGGATCTGACGCCG
aminotransferase      GTCGCCCCAATCCCGATACTTTCCCATTTAACTCGAT
[Cryptococcus         TACCTTAAATTTGAAACCACCTTTAGGCTTGCCCGAG
deuterogattii R265],  AGTTCAAATAACATGCCGGTCTCTATCACGATTGAA
codon optimized for   GACCCCGATTTAGCGACGGCCTTACAATATGCACCT
expression in E. coli AGCGCCGGTATTCCTAAGCTGCGCGAATGGCTGGCT
SEQ ID NO: 171        GACTTACAAGCTCACGTTCATGAGCGCCCCCGTGGC
                      GATTATGCCATCTCGGTCGGGTCGGGGTCACAGGAT
                      TTGATGTTTAAGGGCTTCCAAGCTGTCTTGAATCCAG
                      GTGATCCAGTCCTTCTGGAAACCCCAATGTATTCAGG
                      TGTTCTGCCAGCGCTGCGCATTCTGAAGGCGGATTAT
                      GCAGAAGTTGATGTAGACGACCAGGGGTTATCTGCT
                      AAAAACCTTGAAAAAGTTTTATCAGAGTGGCCCGCA
                      GATAAGAAGCGTCCTCGTGTCCTGTATACGTCGCCA
                      ATCGGCTCCAATCCTTCCGGATGTTCAGCATCCAAGG
                      AACGCAAGTTAGAGGTACTGAAAGTCTGTAAGAAGT
                      ACGATGTGCTGATCTTCGAAGACGATCCGTATTATTA
                      CCTTGCTCAAGAGCTTATTCCATCCTATTTTGCGTTG
                      GAAAAACAAGTTTATCCGGAGGGTGGGCACGTTGTA
                      CGCTTTGACTCATTTAGTAAATTGCTTTCTGCTGGGA
                      TGCGCTTGGGATTTGCTACAGGGCCGAAGGAAATTC
                      TTCATGCGATTGACGTCAGTACAGCAGGCGCAAATT
                      TACATACTTCAGCGGTCTCTCAAGGTGTCGCTCTTCG
                      CCTGATGCAGTATTGGGGGATCGAGGGATTCCTTGC
                      ACATGGCCGCGCGGTGGCCAAACTTTACACGGAGCG
                      CCGCGCTCAGTTCGAGGCAACCGCACATAAGTACCT
                      GGACGGGCTGGCCACTTGGGTATCTCCCGTAGCGGG
                      AATGTTTTTATGGATCGATCTTCGTCCAGCAGGAATC
                      GAAGATTCTTACGAATTAATTCGCCATGAAGCATTA
                      GCCAAAGGCGTTTTAGGCGTTCCAGGGATGGCGTTTT
                      ATCCGACAGGCCGTAAGTCTTCCCATGTTCGTGTCAG
                      TTTCAGTATCGTCGACCTGGAAGACGAATCTGACCTT
                      GGTTTTCAACGCCTGGCTGAAGCTATTAAGGATAAA
                      CGCAAGGCTTTAGGGCTGGCT
Tryptophan            ATGAGTCAAGTGATTAAGAAGAAACGTAACACCTTT
Decarboxylase (EC     ATGATCGGAACGGAGTACATTCTTAACAGTACACAA
4.1.1.28) Chain A,    TTGGAGGAAGCGATTAAATCATTCGTACATGATTTCT
Ruminococcus          GCGCAGAGAAGCATGAGATCCATGATCAACCTGTGG
Gnavus Tryptophan     TAGTAGAAGCTAAAGAACATCAGGAGGACAAAATC
Decarboxylase Rum     AAACAAATCAAAATCCCGGAAAAGGGACGTCCTGTA
gna_01526 (alpha-     AATGAAGTCGTTTCTGAGATGATGAATGAAGTGTAT
fmt); codon           CGCTACCGCGGAGACGCCAACCATCCTCGCTTTTTTT
optimized for the     CTTTTGTGCCCGGACCTGCAAGGAGTGTGTCGTGGTT
expression in E. coli GGGGGATATTATGACGTCCGCCTACAATATrCATGCT
                      GGAGGCTCAAAGCTGGCACCGATGGTTAACTGCATT
                      GAGCAGGAAGTTCTGAAGTGGTTAGCAAAGGAAGTG
                      GGGTTCACAGAAAATCCAGGTGGCGTATTTGTGTCG
                      GGCGGTTCAATGGCGAATATTACGGCACTTACTGCG
                      GCTCGTGACAATAAACTGACCGACATTAACCTTCATT
                      TGGGAACTGCTTATATTAGTGACCAGACTCATAGTTC
                      AGTTGCGAAAGGATTACGCATTATTGGAATCACTGA
                      CAGTCGCATCCGTCGCATTCCCACTAACTCCCACTTC
                      CAGATGGATACCACCAAGCTGGAGGAAGCCATCGAG
                      ACCGACAAGAAGTCTGGCTACATTCCGTTCGTCGTTA
                      TCGGAACAGCAGGTACCACCAACACTGGTTCGATTG
                      ACCCCCTGACAGAAATCTCTGCGTTATGTAAGAAGC
                      ATGACATGTGGTTTCATATCGACGGAGCGTATGGAG
                      CTAGTGTTCTGCTGTCACCTAAGTACAAGAGCCTTCT
                      TACCGGAACCGGCTTGGCTGACAGTATTTCGTGGGA
                      TGCTCATAAATGGTTGTTCCAAACGTACGGCTGTGCA
                      ATGGTACTTGTCAAAGATATCCGTAATTTATTCCACT
                      CTTTTCATGTGAATCCCGAGTATCTTAAGGATCTGGA
                      AAACGACATCGATAACGTTAATACATGGGACATCGG
                      CATGGAGCTGACGCGCCCTGCACGCGGTCTTAAATT
                      GTGGCTTACTTTACAGGTCCTTGGATCTGACTTGATT
                      GGGAGTGCCATTGAACACGGTTTCCAGCTGGCAGTT
                      TGGGCTGAGGAACCATTGAATCCAAAGAAAGACTGG
                      GAGATCGTTTCTCCAGCTCAGATGGCTATGATTAATT
                      TCCGTTATGCCCCTAAGGATTTAACCAAAGAGGAAC
                      AGGATATTCTGAATGAAAAGATCTCCCACCGCATTTT
                      AGAGAGCGGATACGCTGCAATTTTCACTACTGTATTA
                      AACGGCAAGACCGTTTTACGCATCTGTGCAATTCACC
                      CGGAGGCAACTCAAGAGGATATGCAACACAATCG
                      ACTTATTAGACCAATACGGTCGTGAAATCTATACCG
                      AGATGAAGAAAGCG

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 16B or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as listed in Table 16A or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 16B or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide the polypeptide sequences listed in Table 16A or a functional fragment thereof.

In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. Accordingly, in one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 141. In yet another embodiment the Tryptophan Decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 141.

In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. Accordingly, in one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 149. In yet another embodiment the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 149.

In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 150. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 150.

In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 154. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 154.

In one embodiment, genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 85% identity with the entire sequence of one or more sequence(s) of Table 16A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 90% identity with the entire sequence of one or more sequence(s) of Table 16A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 95% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 96%, 97%, 98%, or 99%identity with the entire sequence of one or more sequence(s) of Table 16A. Accordingly, in one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which comprises the entire sequence of one or more sequence(s) of Table 16A.

In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In som embodiments the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

Table 17 depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by indole-3-propionate producing bacteria.

TABLE 17
Non-limiting Examples of Sequences for indole-3-propionate Production
Description      Sequence
FldA: indole-3-  MENNTNMFSGVKVIELANFIAAPAAGRFFADGGAEVIKIESPA
propionyl-       GDPLRYTAPSEGRPLSQEENTTYDLENANKKAIVLNLKSEKGK
CoA: indole-3-   KILHEMLAEADILLTNWRTKALVKQGLDYETLKEKYPKLVFA
lactate CoA      QITGYGEKGPDKDLPGFDYTAFFARGGVSGTLYEKGTVPPNV
transferase from VPGLGDHQAGMFLAAGMAGALYKAKTTGQGDKVTVSLMHS
Clostridium      AMYGLGIMIQAAQYKDHGLVYPINRNETPNPFIVSYKSKDDYF
sporogenes       VQVCMPPYDVFYDRFMTALGREDLVGDERYNKIENLKDGRA
SEQ ID NO: 173   KEVYSIIEQQMVTKTKDEWDKIFRDADIPFAIAQTWEDLLEDE
                 QAWANDYLYKMKYPTGNERALVRLPVFFKEAGLPEYNQSPQI
                 AENTVEVLKEMGYTEQEIEELEKDKDIMVRKEK
FldB: subunit of MSDRNKEVKEKKAKHYLREITAKHYKEALEAKERGEKVGWC
indole-3-lactate ASNFPQEIATTLGVKVVYPENHAAAVAARGNGQNMCEHAEA
dehydratase from MGFSNDVCGYARVNLAVMDIGHSEDQPIPMPDFVLCCNNICN
Clostridium      QMIKWYEHIAKTLDIPMILIDIPYNTENTVSQDRIKYIRAQFDD
sporogenes       AIKQLEEITGKKWDENKFEEVMKISQESAKQWLRAASYAKYK
SEQ ID NO: 174   PSPFSGFDLFNHMAVAVCARGTQEAADAFKMLADEYEENVKT
                 GKSTYRGEEKQRILFEGIACWPYLRHKLTKLSEYGMNVTATV
                 YAEAFGVIYENMDELMAAYNKVPNSISFENALKMRLNAVTST
                 NTEGAVIHINRSCKLWSGFLYELARRLEKETGIPVVSFDGDQA
                 DPRNFSEAQYDTRIQGLNEVMVAKKEAE
FldC: subunit of MSNSDKFFNDFKDIVENPKKYIMKHMEQTGQKAIGCMPLYTP
indole-3-lactate EELVLAAGMFPVGVWGSNTELSKAKTYFPAFICSILQTTLENA
dehydratase from LNGEYDMLSGMMITNYCDSLKCMGQNFKLTVENIEFIPVTVPQ
Clostridium      NRKMEAGKEFLKSQYKMNIEQLEKISGNKITDESLEKAIEIYDE
sporogenes       HRKVMNDFSMLASKYPGIITPTKRNYVMKSAYYMDKKEHTE
SEQ ID NO: 175   KVRQLMDEIKAIEPKPFEGKRVITTGIIADSEDLLKILEENNIAIV
                 GDDIAHESRQYRTLTPEANTPMDRLAEQFANRECSTLYDPEKK
                 RGQYIVEMAKERKADGIIFFMTKFCDPEEYDYPQMKKDFEEA
                 GIPHVLIETDMQMKNYEQARTAIQAFSETL
FldD: indole-3-  MFFTEQHELIRKLARDFAEQEIEPIADEVDKTAEFPKEIVKKMA
acrylyl-CoA      QNGFFGIKMPKEYGGAGADNRAYVTIMEEISRASGVAGIYLSS
reductase from   PNSLLGTPFLLVGTDEQKEKYLKPMIRGEKTLAFALTEPGAGS
Clostridium      DAGALATTAREEGDYYILNGRKTFITGAPISDNIIVFAKTDMSK
sporogenes       GTKGITTFIVDSKQEGVSFGKPEDKMGMIGCPTSDIILENVKVH
SEQ ID NO: 176   KSDILGEVNKGFITAMKTLSVGRIGVASQALGIAQAAVDEAVK
                 YAKQRKQFNRPIAKFQAIQFKLANMETKLNAAKLLVYNAAYK
                 MDCGEKADKEASMAKYFAAESAIQIVNDALQIHGGYGYIKDY
                 KIERLYRDVRVIAIYEGTSEVQQMVIASNLLK
FldH1: indole-3- MKILAYCVRPDEVDSFKKFSEKYGHTVDLIPDSFGPNVAHLAK
lactate          GYDGISILGNDTCNREALEKIKDCGIKYLATRTAGVNNIDFDA
dehydrogenase    AKEFGINVANVPAYSPNSVSEFTIGLALSLTRKIPFALKRVELN
from Clostridium NFALGGLIGVELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGY
sporogenes       DIFENEEAKKYIEYKSLDEVFKEADIITLHAPLTDDNYHMIGKE
SEQ ID NO: 177   SIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEY
                 EQGVFHNNKMNEIMQDDTLERLKSFPNVVITPHLGFYTDEAVS
                 NMVEITLMNLQEFELKGTCKNQRVCK
FldH2: indole-3- MKILMYSVREHEKPAIKKWLEANPGVQIDLCNNALSEDTVCK
lactate          AKEYDGIAIQQTNSIGGKAVYSTLKEYGIKQIASRTAGVDMIDL
dehydrogenase    KMASDSNILVTNVPAYSPNAIAELAVTHTMNLLRNIKTLNKRI
from Clostridium AYGDYRWSADLIAREVRSVTVGVVGTGKIGRTSAKLFKGLGA
sporogenes       NVIGYDAYPDKKLEENNLLTYKESLEDLLREADVVTLHTPLLE
SEQ ID NO: 178   STKYMINKNNLKYMKPDAFIVNTGRGGIINTEDLIEALEQNKIA
                 GAALDTFENEGLFLNKVVDPTKLPDSQLDKLLKMDQVLITHH
                 VGFFTTTAVQNIVDTSLDSVVEVLKTNNSVNKVN
AcuI: acrylyl-   MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYK
CoA reductase    DALAITGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVIL
from Rhodobacter NGWGVGEKHWGGLAERARVRGDWLVPLPAPLDLRQAAMIG
sphaeroides      TAGYTAMLCVLALERHGVVPGNGEIVVSGAAGGVGSVATTLL
SEQ ID NO: 179   AAKGYEVAAVTGRASEAEYLRGLGAASVIDRNELTGKVRPLG
                 QERWAGGIDVAGSTVLANMLSMMKYRGVVAACGLAAGMDL
                 PASVAPFILRGMTLAGVDSVMCPKTDRLAAWARLASDLDPAK
                 LEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP

In one embodiment, the tryptophan pathway catabolic enzyme encoded by the genetically engineered bacteria has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. Accordingly, in one embodiment, the tryptophan pathway catabolic enzyme has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179.

In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g. FIG. 39, FIG. 45A and FIG. 45B. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the al teast one sequence(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits.

Tryptophan Repressor (TrpR)

In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Tryptophan and Tryptophan MetaboliteTransport

Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.

The inner membrane protein YddG of E. coli , encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

In some embodiments, the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.

In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporte gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.

In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.

Inhibitory and Targeting Molecules

In some embodiments, the genetically engineered bacteria of the invention are capable of producing a molecule that is capable of inhibiting a metabolic disease-promoting molecule. The genetically engineered bacteria may express any suitable inhibitory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is capable of neutralizing one or more metabolic disease-promoting molecules, e.g., dipeptidyl peptidase-4 (DPP-4) or ghrelin receptor. The genetically engineered bacteria may inhibit one or more metabolic disease-promoting molecules.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in plants and animals. RNAi is activated when microRNA (miRNA), double-stranded RNA (dsRNA), or short hairpin RNA (shRNA) is processed into short interfering RNA (siRNA) duplexes (Keates et al., 2008). RNAi can be “activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver shRNA to target cells” such as mammalian cells (Keates et al., 2008). In some embodiments, the genetically engineered bacteria of the invention induce RNAi-mediated gene silencing of one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.In some embodiments, the genetically engineered bacteria produce siRNA targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Single-chain variable fragments (scFv) are “widely used antibody fragments . . . produced in prokaryotes” (Frenzel et al., 2013). scFv lacks the constant domain of a traditional antibody and expresses the antigen-binding domain as a single peptide. Bacteria such as Escherichia coli are capable of producing scFv that target a variety of molecules, e.g., TNF (Hristodorov et al., 2014). In some embodiments, the genetically engineered bacteria of the invention express a binding protein for neutralizing one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria produce scFv targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria produce both scFv and siRNA targeting one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose (see, e.g., Xiao et al., 2014).

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Generation of Bacterial Strains with Enhanced Ability to Transport Amino Acids

Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

For example, if the biosynthetic pathway for producing an amino acid is disrupted a strain capable of high-affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid—at growth-limiting concentrations—will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid.

Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate.

Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations “screened” throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 1011.2 CCD1. This rate can be accelerated by the addition of chemical mutagens to the cultures—such as N-methyl-N-nitro-N-nitrosoguanidine (NTG)—which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. Ø. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

Similar methods can be used to generate E. Coli Nissle mutants that consume or import tryptophan and/or kynurenine.

Regulation of Payload Expression

In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.

Herein the term “payload” refers to one or more effector molecules described herein and/or one or more enzyme(s) or polypeptide(s0 needed for the production of such effector molecules. Non-limiting examples of payloads include butyrate, propionate, acetate, and butyrate and/or propionate and/or acetate producing enzymes,. Further examples include GLP-1, GLP-2, manganese transporters, GABA transporters, tryptophan and/pr tryptophan metabolite transporters, aromatic amino acid transporters, and polypeptides for metabolizing (catabolizing) GABA. Yet further examples include tryptophan and/or one or more of its metabolites, e.g., KP metabolites ann/or indole metabolites as described herein, and/or one or more enzyme(s) for the production of tryptophan and/or one or more of its metabolites, and/or one or more gut-barrier enhancing molecule(s) and/or antinflammatory molecules described herein. Yet other examples include bile salt hydrolases, bile salte transporters, and bile salt exporters described herein.

In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is induced under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manuafacturing conditions). In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manuafacturing conditions).

In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. . In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell.

FNR dependent Regulation

The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule, wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the inducible promoter is an oxygen level-dependent promoter and the metabolic and/or satiety effector and/or immune modulator molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level-sensing transcription factor, thereby driving production of the metabolic and/or satiety effector and/or immune modulator molecule

Bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An oxygen level-dependent promoter is a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. In one embodiment, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter. In a more specific aspect, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.

In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In E. coli , FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in Table 18 and Table 19 below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.

TABLE 18
FNR Promoter Sequences
FNR Responsive
Promoter       Sequence
SEQ ID NO: 180 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA
               CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT
               CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA
               GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC
               TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT
               GCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA
SEQ ID NO: 181 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGG
               CTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAA
               TATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCG
               ACTGTAAATCAGAAAGGAGAAAACACCT
SEQ ID NO: 182 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA
               CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT
               CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA
               GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC
               TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT
               GCTGAATCGTTAAGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAG
               AAGGAGATATACAT
SEQ ID NO: 183 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATG
               GCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAA
               ATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCCCTCTAGA
               AATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
SEQ ID NO: 184 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGG
               TTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTA
               AAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCT
               CTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAT
               ATACAT

TABLE 19
FNR Promoter sequences
FNR-responsive
regulatory region 12345678901234567890123456789012345678901234567890
SEQ ID NO: 185    ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTA
                  GAGCGTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATC
                  AGAGGGCCGACAGGCTCCCACAGGAGAAAACCG
SEQ ID NO: 186    CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTAC
                  CTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCG
                  ACAGGCTCCCACAGGAGAAAACCG
nirB1             GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGG
SEQ ID NO: 187    GCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTA
                  CGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTT
                  GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA
                  ATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAA
                  AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC
                  GTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA
nirB2             CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGC
SEQ ID NO: 188    TTACAGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCT
                  TCCTGTTAGGTTTCGTCAGCCGTCACCGTCAGCATAACACCC
                  TGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCG
                  TCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCT
                  ATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAA
                  TATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCA
                  GCAATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTT
                  ACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGT
                  AATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaaggagatat
                  acat
nirB3             GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGG
SEQ ID NO: 189    ACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTA
                  CGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTT
                  GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA
                  ATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAA
                  AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC
                  GTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA
ydfZ              ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGA
SEQ ID NO: 190    CTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATC
                  AAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTG
                  CGCCCGTTACGTGGGCTTCGACTGTAAATCAGAAAGGAGAA
                  AACACCT
nirB + RBS        GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGG
SEQ ID NO: 191    GCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTA
                  CGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTT
                  GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA
                  ATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAA
                  AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC
                  GTTAAGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA
                  GGAGATATACAT
ydfZ + RBS        CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCG
SEQ ID NO: 192    ACTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGA
                  TCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATT
                  GCGCCCGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGA
                  AGGAGATATACAT
fnrS1             AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT
SEQ ID NO: 193    AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC
                  AAAAACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTA
                  CCCATTCAGGGCAATATCTCTCTTGGATCCCTCTAGAAATA
                  ATTTTGTTTAACTTTAAGAAGGAGATATACAT
fnrS2             AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT
SEQ ID NO: 194    AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC
                  AAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTA
                  CCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAACTCT
                  AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
nirB + crp        TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCA
SEQ ID NO: 195    CCGTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCC
                  GGACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCG
                  CTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTT
                  TTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAG
                  AAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATA
                  TAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGA
                  ATCGTTAAGGTAGaaatgtgatctagttcacatttGCGGTAATAGAAAAG
                  AAATCGAGGCAAAAatgtttgtttaactttaagaaggagatatacat
fnrS + crp        AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT
SEQ ID NO: 196    AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC
                  AAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTA
                  CCCATTCAGGGCAATATCTCTCaaatgtgatctagttcacattttttgtttaacttta
                  agaaggagatatacat

FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.

As used herein the term “payload” refers to one or more e.g. anti-inflammation and/or gut barrier function enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, GLP1, and/or tryptophan and/or its metabolites. As used herein, the term “polypeptide of interest” or “polypeptides of interest”, “protein of interest”, “proteins of interest”, “payload”, “payloads” further includes any or a plurality of any of the short chain fatty acid producing enzymes, trypophan synthesis, tryptophan metabolite producing enzymes, or bile salt hydrolases and/or bile salt transporters or exporters, enzymes producing any gut barrier enhancer and/or anti-inflammatory metabolite, metabolite transporters or exporters, described herein. As used herein, the term “gene of interest” or “gene sequence of interest” includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more anti-inflammation and/or gut barrier function enhancer molecule(s) described herein.

Non-limiting FNR promoter sequences are provided in Table 6. Table 6 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, nirB1 promoter (SEQ ID NO: 187), nirB2 promoter (SEQ ID NO: 188), nirB3 promoter (SEQ ID NO: 189), ydfZ promoter (SEQ ID NO: 190), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 191), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 192), fnrS, an anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 193 or fnrS2 promoter SEQ ID NO: 194), nirB promoter fused to a crp binding site (SEQ ID NO: 195), and fnrS fused to a crp binding site (SEQ ID NO: 196).

In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of anaerobic regulation of arginine deiminiase and nitrate reduction transcriptional regulator (ANR). In P. aeruginosa, ANR is “required for the expression of physiological functions which are inducible under oxygen-limiting or anaerobic conditions” (Winteler et al., 1996; Sawers 1991). P. aeruginosa ANR is homologous with E. coli FNR, and “the consensus FNR site (TTGAT----ATCAA) was recognized efficiently by ANR and FNR” (Winteler et al., 1996). Like FNR, in the anaerobic state, ANR activates numerous genes responsible for adapting to anaerobic growth. In the aerobic state, ANR is inactive. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all have functional analogs of ANR (Zimmermann et al., 1991). Promoters that are regulated by ANR are known in the art, e.g., the promoter of the arcDABC operon (see, e.g., Hasegawa et al., 1998).

In other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Görke and Stülke, 2008). In some embodiments, the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g., an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro.

In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen-level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006). In some embodiments, both the oxygen level-sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.

In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid.

In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.

RNS-Dependent Regulation

In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.

As used herein, “reactive nitrogen species” and “RNS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO●), peroxynitrite or peroxynitrite anion (ONOO—), nitrogen dioxide (●NO2), dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO2—) (unpaired electrons denoted by ●). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

As used herein, “RNS-inducible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s), e.g., any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

As used herein, “RNS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

As used herein, “RNS-repressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

As used herein, a “RNS-responsive regulatory region” refers to a RNS-inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 20.

TABLE 20
Examples of RNS-sensing transcription
factors and RNS-responsive genes
		Examples of responsive genes,
RNS-sensing	Primarily capable	promoters, and/or regulatory
transcription factor:	of sensing:	regions:
NsrR	NO	norB, aniA, nsrR, hmpA, ytfE,
		ygbA, hcp, hcr, nrfA, aox
NorR	NO	norVW, norR
DNR	NO	norCB, nir, nor, nos

1.

In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR “is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide” (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload(s).

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) “promotes the expression of the nir, the nor and the nos genes” in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of the dissimilatory nitrate respiration regulator (DNR). DNR is a member of the FNR family (Arai et al., 1995) and is a transcriptional regulator that is required in conjunction with ANR for “anaerobic nitrate respiration of Pseudomonas aeruginosa” (Hasegawa et al., 1998). For certain genes, the FNR-binding motifs “are probably recognized only by DNR” (Hasegawa et al., 1998). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used. Non-limiting examples include ArcA/B, ResD/E, NreA/B/C, and AirSR, and others are known in the art.

In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is “an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).

In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a payload. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments, include, but are not limited to, TetR, C1, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a payload gene or genes is expressed.

A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).

In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.

In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).

In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of payload in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.

ROS-Dependent Regulation

In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

As used herein, “reactive oxygen species” and “ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal-catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H2O2), organic peroxide (ROOH), hydroxyl ion (OH—), hydroxyl radical (●OH), superoxide or superoxide anion (●O2-), singlet oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl radical (●O2-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOC1), nitric oxide (NO●), and peroxynitrite or peroxynitrite anion (ONOO—) (unpaired electrons denoted by ●). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014).

As used herein, “ROS-inducible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

As used herein, “ROS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

As used herein, “ROS-repressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

As used herein, a “ROS-responsive regulatory region” refers to a ROS-inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 21.

TABLE 21
Examples of ROS-sensing transcription factors
and ROS-responsive genes
ROS-sensing		Examples of responsive genes,
transcription	Primarily capable	promoters, and/or regulatory
factor:	of sensing:	regions:
OxyR	H2O2	ahpC; ahpF; dps; dsbG; fhuF; flu;
		fur; gor; grxA; hemH; katG; oxyS;
		sufA; sufB; sufC; sufD; sufE; sufS;
		trxC; uxuA; yaaA; yaeH; yaiA; ybjM;
		ydcH; ydeN; ygaQ; yljA; ytfK
PerR	H2O2	katA; ahpCF; mrgA; zoaA; fur;
		hemAXCDBL; srfA
OhrR	Organic peroxides	ohrA
	NaOCl
SoxR	•O2−	soxS
	NO•
	(also capable of
	sensing H2O2)
RosR	H2O2	rbtT; tnp16a; rluC1; tnp5a; mscL;
		tnp2d; phoD; tnp15b; pstA; tnp5b;
		xylC; gabD1; rluC2; cgtS9; azlC;
		narKGHJI; rosR

2.

In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.

In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR “functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g., “genes involved in H2O2 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)” and “OxyS, a small regulatory RNA” (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene. In the presence of ROS, e.g., H2O2, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS-inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is “activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression” (Koo et al., 2003). “SoxR is known to respond primarily to superoxide and nitric oxide” (Koo et al., 2003), and is also capable of responding to H2O2. The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al., 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload.

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR “binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event,” but oxidized OhrR is “unable to bind its DNA target” (Duarte et al., 2010). OhrR is a “transcriptional repressor [that] . . . senses both organic peroxides and NaOC1” (Dubbs et al., 2012) and is “weakly activated by H2O2 but it shows much higher reactivity for organic hydroperoxides” (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a payload gene. In the presence of ROS, e.g., NaOC1, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload.

OhrR is a member of the MarR family of ROS-responsive regulators. “Most members of the MarR family are transcriptional repressors and often bind to the −10 or −35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012).

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is “a MarR-type transcriptional regulator” that binds to an “18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is “reversibly inhibited by the oxidant H2O2” (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to “a putative polyisoprenoid-binding protein (cg1322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cg1426), two putative FMN reductases (cg1150 and cg1850), and four putative monooxygenases (cg0823, cg1848, cg2329, and cg3084)” (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload. In the presence of ROS, e.g., H2O2, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.

In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR “when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)” (Marinho et al., 2014). PerR is a “global regulator that responds primarily to H202” (Dubbs et al., 2012) and “interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes” (Marinho et al., 2014). PerR is capable of binding a regulatory region that “overlaps part of the promoter or is immediately downstream from it” (Dubbs et al., 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al., 2012).

In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.

A ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although “OxyR is primarily thought of as a transcriptional activator under oxidizing conditions . . . OxyR can function as either a repressor or activator under both oxidizing and reducing conditions” (Dubbs et al., 2012), and OxyR “has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)” (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by RosR. In addition, “PerR-mediated positive regulation has also been observed . . . and appears to involve PerR binding to distant upstream sites” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by PerR.

One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 22. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, or SEQ ID NO: 200, or a functional fragment thereof.

TABLE 22
Nucleotide sequences of exemplary OxyR-regulated regulatory
regions
Regulatory
sequence Sequence
katG     TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA
(SEQ ID  GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT
NO: 197) TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA
         AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA
         AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA
         ACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAATTCAA
         TTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTGTA
         GAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAAGGT
         ACC
dps      TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT
(SEQ ID  CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG
NO: 198) CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCGGA
         ACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACA
         TAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGAGA
         AAGGTACC
ahpC     GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC
(SEQ ID  CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG
NO: 199) CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA
         CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC
         AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTTA
         TCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAATTG
         GTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATGCG
         AATTCATTAAAGAGGAGAAAGGTACC
oxyS     CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGCG
(SEQ ID  ATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTCTG
NO: 200) ACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGTA
         CC

In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS-responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.

In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS-responsive regulatory region.

Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.

In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome.

Propionate and Other Promoters

In some embodiments, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., the mammalian gut. For example, the short-chain fatty acid propionate is a major microbial fermentation metabolite localized to the gut (Hosseini et al., 2011). In one embodiment, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is under the control of a propionate-inducible promoter that is activated by the presence of propionate in the mammalian gut. Any molecule or metabolite found in the mammalian gut, in a healthy and/or disease state, may be used to induce payload expression. Non-limiting examples of inducers include propionate, bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese. In alternate embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule and/or immune modulator is under the control of a pBAD promoter, which is activated in the presence of the sugar arabinose.

In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule, such that the gene or gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, gene or gene cassette for producing the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed on a chromosome.

Table 23 lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut.

TABLE 23
Propionate promoter sequence
Description      Sequence
Prp (Propionate) TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCA
promoter         CAGCGTGGTACGGCTGATCCCCAAATAACGTGCGGCGGCGCG
Bold: prpR       CTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG
Lower case:      CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCAC
ribosome binding CAGTTCAGGTAATAATTGCCGCATAAATTGTCTGTCCAGTGT
site             TGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT
ATG underlined:  CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTT
start of gene of CAGTAGAAGCGGCTGACACTGCGTCAGCCCATGACGCACCGA
interest         TTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA
SEQ ID NO: 201   AAAGTTTTCCGCCAGAGGCAGAATATCAGGCTGTCGCTCGCG
                 CAAGGGGGGAAGCGGCAGACGCAGAATGCTCAAACGGTAAAA
                 CAGATCGGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG
                 ATCGCAATGCGTGGCGCTGATCACCCGGACATCTACCGGGAT
                 CGGCTGATGCCCGCCAACGCGGGTGACGGCTTTTTCCTCCAG
                 TACGCGTAGAAGGCGGGTTTGTAACGGCAGCGGCATTTCGCC
                 AATTTCGTCAAGAAACAGCGTGCCGCCGTGGGCGACCTCAAA
                 CAGCCCCGCACGTCCACCTCGTCTTGAGCCGGTAAACGCTCC
                 CTCCTCATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGGT
                 AATCGCGCCGCAATTAACGGCGACAAAGGGCGGAGAAGGCTT
                 GTTCTGACGGTGGGGCTGACGGTTAAACAACGCCTGATGAAT
                 CGCTTGCGCCGCCAGCTCTTTCCCGGTCCCTGTTTCCCCCTG
                 AATCAGCACTGCCGCGCGGGAACGGGCATAGAGTGTAATCGT
                 ATGGCGAACCTGCTCCATTTGTGGTGAATCGCCGAGGATATC
                 GCTCAGCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTATG
                 CTGGCTATACTGACGCCGTGTCAGGCGGGTCATATCCAGCGC
                 ATCATGGAAAGCCTGACGTACGGTGGCCGCTGAATAAATAAA
                 GATGGCGGTCATTCCTGCCTCTTCCGCCAGGTCGGTAATTAG
                 TCCTGCCCCAATTACAGCCTCAATGCCGTTAGCTTTGAGCTC
                 GTTAATTTGCCCGCGAGCATCCTCTTCAGTGATATAGCTTCG
                 CTGTTCAAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG
                 AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGA
                 AGTCAGCTTTCCCGCTTTTGCCAGAGCCTGTAATACATCGAA
                 TCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT
                 TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTC
                 GCAGCGTTCGGTTGCCAGTTTTTTGCGAATGTAGGCTACTGC
                 CTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG
                 ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCG
                 CGTTACCGAGACCGTCCAGATCACCGGTTTATCGCTATTATC
                 GCGCGAAGCGCTATGCACAGTAACCATCGTCGTAGATTCATG
                 TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGT
                 TGCAATGAAACACAGGCCTCCGTTTCATGAAACGTTAGCTGA
                 CTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT
                 TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATG
                 GTTTCTCGGTTTTTGGGTCTGGCATATCCCTTGCTTTAATGA
                 GTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag
                 taatttcaacaaaataacgagcattcgaatg

Other Inducible Promoters

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an arabinose inducible system. The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub-saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L-arabinose transporter (Zhang et al., Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ. Microbiol. August 2012 vol. 78 no. 16 5831-5838). Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in FIG. 79.

In one embodiment, expression of one or more protein(s) of interest, e.g., one or more therapeutic polypeptide(s), is driven directly or indirectly by one or more arabinose inducible promoter(s).

In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., arabinose.

In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown arerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically.

In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 202. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 203. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 204.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in FIG. 82B (construct for PAL expression under the control of a rhamnose inducible promoter).

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.

In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., rhamnose

In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically.

In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 205.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydrolyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacI) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli , its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate LacI, can be used instead of IPTG in a similar manner.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s).

In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., IPTG.

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.

In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 206. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacI, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 207. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 208.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M & Bujard H. PNAS. 1992 Jun. 15; 89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline-controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus.In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline-controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a tetracycline inducible promoter.

In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., tetracycline

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.

In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 213 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 213 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 213 in italics (Tet repressor is in italics).

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun. 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant c1857 repressor and the pL and/or pR phage promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the 2\, promoters can then be efficiently regulated by the mutant thermolabile c1857 repressor of bacteriophage λ. At temperatures below 37° C., cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in FIG. 82A. Inducible expression from the ParaBad can be controlled or further fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a thermoregulated promoter.

In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature.

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.

In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabino se). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 209. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 210. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 212.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.

This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. FIG. 83A depicts a schematic of the gene organization of a PssB promoter.

In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s).

In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest. In some embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.

In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 214.

Sequences useful for expression from inducible promoters are listed in Table 24.

TABLE 24
Inducible promoter construct sequences
Description      Sequence
Arabinose        CAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC
Promoter region  TAACCCAACCGGTAACCCCGCTTATTAAAAGCATTCTGTA
SEQ ID NO:       ACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAA
202              AAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTAT
                 TTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATC
                 CATAAGATTAGCGGATCCAGCCTGACGCTTTTTTTCGCAA
                 CTCTCTACTGTTTCTCCATACCTCTAGAAATAATTTTGTTT
                 AACTTTAAGAAGGAGATATACAT
AraC (reverse    TTATTCACAACCTGCCCTAAACTCGCTCGGACTCGCCCCG
orientation)     GTGCATTTTTTAAATACTCGCGAGAAATAGAGTTGATCGT
SEQ ID NO:       CAAAACCGACATTGCGACCGACGGTGGCGATAGGCATCC
203              GGGTGGTGCTCAAAAGCAGCTTCGCCTGACTGATGCGCTG
                 GTCCTCGCGCCAGCTTAATACGCTAATCCCTAACTGCTGG
                 CGGAACAAATGCGACAGACGCGACGGCGACAGGCAGACA
                 TGCTGTGCGACGCTGGCGATATCAAAATTACTGTCTGCCA
                 GGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATT
                 ATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATG
                 CGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCA
                 ATTCCGAATAGCGCCCTTCCCCTTGTCCGGCATTAATGATT
                 TGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCAT
                 CCGGGCGAAAGAAACCGGTATTGGCAAATATCGACGGCC
                 AGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGT
                 AAACCCACTGGTGATACCATTCGTGAGCCTCCGGATGACG
                 ACCGTAGTGATGAATCTCTCCAGGCGGGAACAGCAAAAT
                 ATCACCCGGTCGGCAGACAAATTCTCGTCCCTGATTTTTCA
                 CCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACC
                 TTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAA
                 CCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGG
                 CGTTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGC
                 TTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAA
                 ACCAATTGTCCATATTGCAT
AraC             MQYGQLVSSLNGGSMKSMAEAQNDPLLPGYSFNAHLVAGL
polypeptide      TPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFV
SEQ ID NO:       CRPGDILLFPPGEIHHYGRHPEAHEWYHQWVYFRPRAYWHE
204              WLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYS
                 ELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHL
                 ADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRIS
                 QAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSE
                 FRAGCE*
Region           CGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAAC
comprising       ATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTT
rhamnose         TCCTGTCAGTAACGAGAAGGTCGCGAATCAGGCGCTTTTT
inducible        AGACTGGTCGTAATGAAATTCAGCTGTCACCGGATGTGCT
promoter         TTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTAC
SEQ ID NO:       AAATAATTTTGTTTAAAACAACACCCACTAAGATAACTCT
205              AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
Lac Promoter     ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG
region           CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG
SEQ ID NO:       CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA
206              TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG
                 TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG
                 TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT
                 CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA
                 CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA
                 CAT
LacO             GGAATTGTGAGCGCTCACAATT
LacI (in reverse TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC
orientation)     TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT
SEQ ID NO:       GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA
207              GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA
                 GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA
                 GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA
                 ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG
                 ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC
                 GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT
                 CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT
                 TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT
                 CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG
                 CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA
                 TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG
                 ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG
                 AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC
                 AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC
                 AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC
                 AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG
                 CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC
                 ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG
                 CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG
                 AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG
                 TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC
                 ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG
                 GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG
                 ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT
                 TCAT
LacI             MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEA
polypeptide      AMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAA
sequence         IKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLI
SEQ ID NO:       INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT
208              RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN
                 QIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQ
                 MALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIK
                 QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA
                 PNTQTASPRALADSLMQLARQVSRLESGQ
Region           ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC
comprising       GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG
Temperature      CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCC
sensitive        GTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAA
promoter         CAACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAA
SEQ ID NO:       CTTTAAGAAGGAGATATACAT
209
mutant cI857     TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT
repressor        TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG
SEQ ID NO:       GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT
210              ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCA
                 AGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCT
                 CAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGG
                 CTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACC
                 TCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGC
                 TTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGC
                 TTAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACA
                 GGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAA
                 CCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGG
                 GCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCA
                 ATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAA
                 TAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCT
                 GCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTT
                 TTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGC
                 TCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT
RBS and leader   CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA
region           CAT
SEQ ID NO:
211
mutant cI857     MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM
repressor        GMGQSGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAR
polypeptide      EIYEMYEAVSMQPSLRSEYEYPVFSHVQAGMFSPKLRTFTKG
sequence         DAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGML
SEQ ID NO:       ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP
212              QYPMIPCNESCSVVGKVIASQWPEETFG
TetR-Tet         Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataa
promoter         gaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcata
construct        ctatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacct
SEQ ID NO:       aaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttgg
213              cataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgta
                 cttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaat
                 cttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggct
                 aaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacaccta
                 gcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcg
                 ctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgttgacactctatcattg
                 atagagttattttaccactccctatcagtgatagagaaaagtgaactctagaaataattttgttt
                 aactttaagaaggagatatacat
PssB promoter    tcacctttcccggattaaacgcttttttgcccggtggcatggtgctaccggcgatcacaaacggtta
SEQ ID NO:       attatgacacaaattgacctgaatgaatatacagtattggaatgcattacccggagtgttgtgtaac
214              aatgtctggccaggtttgtttcccggaaccgaggtcacaacatagtaaaagcgctattggtaatgg
                 tacaatcgcgcgtttacacttattc

Constitutive Promoters

In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.

In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoteris active under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

In some embodiments, the constitutive promoterthat is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions).

In some embodiments, the constitutive promoteris active in exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the constitutive promoteris active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoteris active in low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the constitutive promoteris active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoteris directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the constitutive promoteris active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.

Bacterial constitutive promoters are known in the art. For example, a lisitng of suitable promoters from a number of bacterial species and bacteriophages can be found at: http://parts.igem.org/Promoters/Catalog/Constitutive.

Induction of Payloads During Strain Culture

In some embodiments, it is desirable to pre-induce payload or protein of interest expression and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term “bacterial culture” or bacterial cell culture” or “culture” refers to bacterial cells or microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term “fermentation” refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like.

Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. In some embodiments, phenylalanine is added to the media, e.g., to boost cell health. Without wishing to be bound by theory, addition of phenylalanine to the medium may prevent bacteria from catabolizing endogenously produced phenylalanine required for cell growth.

In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is “wasted”, in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites).

In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters.

In some embodiments, the strains are administered without any pre-induction protocols during strain growth prior to in vivo administration.

Anaerobic Induction

In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) and /or Phe transporters under the control of one or more FNR promoters.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.

In one embodiment, expression of two or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under anaerobic or low oxygen conditions.

Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

In one embodiment, expression of one or more Payload is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabino se and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload and or Phe transporter sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Aerobic Induction

In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.

In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s)and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabino se and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload and or Phe transporter sequence(s) under the control of one or more constitutive promoter(s) active under aerobic conditions.

In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload and/or Phe transporter sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.

Microaerobic Induction

In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to “strike a balance” between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) and/or Phe transporter(s) are driven by a anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours.

In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.

Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Induction of Strains Using Phasing, Pulsing and/or Cycling

In some embodiments, cycling, phasing, or pulsing techniques are emplyed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficienty induce and grow the strains prior to in vivo administration. This method is used to “strike a balance” between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g., for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g., reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.

In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.

In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non-limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g., for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1×10̂8 to 1×10̂11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.

In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g., 37 C). In some embodiments, the cells are then induced, e.g., by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload, e.g., PAL.

In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer.

In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques.

Any of the strains described herein can be grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques.

Aerobic Induction of the FNR Promoter

FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, oxygen bypass system shown and described in FIG. 78 is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of the protein of interest (e.g., one or more metabolic effector(s) described herein) by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of the protein of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the protein of interest, e.g., one or more metabolic effector(s) described herein.

In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non-limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g., from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest (e.g., one or more metabolic effector(s) described herein) are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.

In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

Mutagenesis

In some embodiments, an inducible promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, an oxygen level-dependent promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the oxygen level-dependent promoter is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces. In some embodiments, one or more transcription factor binding sites is mutagenized to increase or decrease binding. In alternate embodiments, the wild-type binding sites are left intact and the remainder of the regulatory region is subjected to mutagenesis. In some embodiments, the mutant promoter is inserted into the genetically engineered bacteria of the invention to increase expression of the metabolic and/or satiety effector and/or immune modulator molecule in low-oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the oxygen level-sensing transcription factor and/or the oxygen level-dependent promoter is a synthetic, non-naturally occurring sequence. In some embodiments, the transcription factor regulating the mutated promoter senses the presence of certain molecules or metabolites, the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene encoding a metabolic and/or satiety effector and/or immune modulator molecule is mutated to increase expression and/or stability of said molecule in low oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, one or more of the genes in a gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule is mutated to increase expression of said molecule in low oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions.

Multiple Mechanisms of Action

In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/1, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 57. For example, the genetically engineered bacteria may include four copies of GLP-1 inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of GLP-1 inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three copies of a butyrogenic gene cassette inserted at three different insertion sites, e.g., dapA, cea, and araC/BAD.

In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites and three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites.

In some embodiments, the genetically engineered bacteria comprise one or more of (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein (6) one or more polypetides for secretion, including but not limited to.GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthesis and/or catabolic enzymes of the tryptophan degradation pathways, in wild type or in mutated form (for increased stability or metabolic activity) (3) one or more components of secretion machinery, as described herein (4) one or more auxotrophies, e.g., deltaThyA (5) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (6) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein (7) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway.

In some embodiments, under conditions where the gene, gene(s), or gene cassettes for producing the payload(s) is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload(s) as compared to unmodified bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria of the invention produce at least one metabolic and/or satiety effector and/or immune modulator molecule under inducing conditions and are capable of reducing one or more symptoms of metabolic disease in a subject by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to unmodified bacteria of the same subtype under the same conditions. In some embodiments, under conditions where the payload is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

Symptoms and manifestations of metabolic diseases may be measured by methods known in the art, e.g., glucose tolerance testing, insulin tolerance testing.

In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of a metabolic and/or satiety effector and/or immune modulator molecule under inducing conditions than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the metabolic and/or satiety effector and/or immune modulator molecule. In embodiments using genetically modified forms of these bacteria, the metabolic and/or satiety effector and/or immune modulator molecule will be detectable under inducing conditions.

In certain embodiments, the metabolic and/or satiety effector and/or immune modulator molecule is butyrate. Methods of measuring butyrate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μM/OD, at least about 10 μM/OD, at least about 100 μM/OD, at least about 500 μM/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In certain embodiments, the metabolic and/or satiety effector and/or immune modulator molecule is propionate. Methods of measuring propionate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Hillman 1978; Lukovac et al., 2014). In some embodiments, measuring the activity and/or expression of one or more gene products in the propionate gene cassette serves as a proxy measurement for propionate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure propionate production. In alternate embodiments, propionate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 μM, at least about 10 μM, at least about 100 μM, at least about 500 μM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, or at least about 50 mM of propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the gene, gene(s), or gene cassettes for producing the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload RNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s).

In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s).

Secretion

In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting a molecule from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in FIG. 61, FIG. 62, FIG. 63, FIG. 64, FIG. 65, FIG. 66, and FIG. 67. Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from Shigella, Salmonella, E. coli , Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologouse protein or peptide comprises a type III secretion sequence that allows the molecule of interest o be secreted from the bacteria.

In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173-bp region, is fused to the gene encoding the polypeptide of interest can be used to secrete heterologous polypeptides (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005 Apr.; 23(4):475-81). In some cases, the untranslated region from the fliC loci, may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et al., Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437-7438).

In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in FIG. 62, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex (Teta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. FIG. 63 shows the alpha-hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane-spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli ), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides)—particularly those of eukaryotic origin—contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

One way to secrete properly folded proteins in gram-negative bacteria—particularly those requiring disulphide bonds—is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These “leaky” gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a “leaky” or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, 1 pp, ompC, ompA, ompF, tolA, to1B, pal, degS, degP, and nlpI. Lpp is the most abundant polypeptide in the bacterial cell existing at ˜500,000 copies per cell and functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. 1.

Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). To1A-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpI can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated 1 pp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, to1B, and pal genes. in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlp1. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from 1pp, ompA, ompF, to1 A, to1B, pal, degS, degP, and nlpl genes.

To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from 1 pp, ompA, ompF, to1A, to1B, pa1, degS, degP, and nlp1, under the control of an inducible promoter. For example, expression of 1 pp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., over expression of colicins or the third topological domain of To1A, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

The Table 25 and Table 26 below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

TABLE 25
Secretion systems for gram positive bacteria
Bacterial Strain               Relevant Secretion System
C. novyi-NT (Gram+)            Sec pathway
                               Twin-arginine (TAT) pathway
C. butryicum (Gram+)           Sec pathway
                               Twin-arginine (TAT) pathway
Listeria monocytogenes (Gram+) Sec pathway
                               Twin-arginine (TAT) pathway

TABLE 26
Secretion Systems for Gram negative bacteria
Protein secretary pathways (SP) in gram-negative bacteria and their descendants
						#
Type						Proteins/	Energy
(Abbreviation)	Name	TC#2	Bacteria	Archaea	Eukarya	System	Source
IMPS - Gram-negative bacterial inner membrane channel-forming translocases
ABC (SIP)	ATP binding	3.A.1	+	+	+	3-4	ATP
	cassette
	translocase
SEC (IISP)	General secretory	3.A.5	+	+	+	~12	GTP OR
	translocase						ATP + PMF
Fla/Path (IIISP)	Flagellum/	3.A.6	+	−	−	>10	ATP
	virulence-
	related translocase
Conj (IVSP)	Conjugation-related	3.A.7	+	−	−	>10	ATP
	translocase
Tat (IISP)	Twin-arginine	2.A.64	+	+	+	2-4	PMF
	targeting				(chloroplasts)
	translocase
Oxa1 (YidC)	Cytochrome oxidase	2.A.9	+	+	+	1	None
	biogenesis family				(mitochondria		or
					chloroplasts)		PMF
MscL	Large conductance	1.A.22	+	+	+	1	None
	mechanosensitive
	channel family
Holins	Holin functional	1.E.1•21	+	−	−	1	None
	superfamily
Eukaryotic Organelles
MPT	Mitochondria 1	3.A.B	−	−	+	>20	ATP
	protein translocase				(mitochondrial)
CEPT	Chloroplast envelope	3.A.9	(+)	−	+	≥3	GTP
	protein translocase				(chloroplasts)
Bcl-2	Eukaryotic Bcl-2	1.A.21	−	−	+	1?	None
	family (programmed
	cell death)
Gram-negative bacterial outer membrane channel-forming translocases
MTB (IISP)	Main terminal	3.A.15	+b	−	−	~14	ATP;
	branch of the						PMF
	general secretory
	translocase
FUP AT-1	Fimbrial	1.B.11	+b	−	−	1	None
	usher protein	1.B.12	+b		−	1	None
	Autotransporter-1
AT-2 OMF (ISP)	Autotransporter-2	1.B.40	+b	−	−	1	None
		1.B.17	+b		+(?)	1	None
TPS Secretin		1.B.20	+	−	+	1	None
(IISP and IISP)		1.B.22	+b		−	1	None
OmpIP	Outer membrane	1.B.33	+	−	+	≥4	None?
	insertion porin				(mitochondria;
					chloroplasts)

The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe/Volume 1, Number 9, 2006 “Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently”, the contents of which is herein incorporated by reference in its entirety.

Any of the secretion systems described herein may according to the disclosure be employed to secrete the proteins of interest. Non-limiting examples of proteins of interest include GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, e.g. in the indole pathway and/or the kynurenine pathway, and bile salt hydrolases, as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity.

TABLE 27
Comparison of Secretion systems for secretion of polypeptide from
engineered bacteria
Secretion
System	Tag	Cleavage	Advantages	Other features
Modified	mRNA	No	No peptide tag	May not be as
Type III	(or N-	cleavage	Endogenous	suited for larger
(flagellar)	terminal)	necessary		proteins Deletion
				of flagellar genes
Type V	N- and	Yes	Large proteins	2-step secretion
autotransport	C-		Endogenous
	terminal		Cleavable
Type I	C-	No		Tag; Exogenous
	terminal			Machinery
Diffusible	N-	Yes	Disulfide bond	May affect cell
Outer	terminal		formation	fragility/
Membrane				survivability/
(DOM)				growth/yield

In some embodiments, the therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, such as, one or more GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, and/or bile salt hydrolases, is assembled behind a fliC-5′UTR (e.g., 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of the therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose is used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing conditions, as described herein and known in the art, can be used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing and in vivo conditions, described herein and known in the art, can be used. In other embodiments, a constitutive promoter can be used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In some embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g., the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.

In some embodiments, the therapeutic polypeptides of interest, e.g., such as, one or more GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, and/or tryptophan catabolic enzymes, and/or bile salt hydrolases, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the therapeutic polypeptide of interest is fused to a Tat-dependent secretion signal. Exemplary Tat-dependent tags include TorA, FdnG, and DmsA. In some embodiments, expression of the secretion-tagged therapeutic protein is driven by a tet promoter or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), or by promoters induced by molecules specific to certain metabolic conditions, or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing conditions, as described herein and known in the art, can be used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing and in vivo conditions, described herein and known in the art, can be used. In other embodiments, a constitutive promoter can be used. In some embodiments, the secretion-tagged therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In other embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into the bacterial chromosome, e.g., at one or more of the integration sites shown in FIG. 57. In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include 1pp, pal, to1A, and/or nlpI. In some embodiments, 1 pp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, to1 A is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non-limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.

In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-1, GLP-1 peptides, GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, SCFA producing enzymes, Tryptophan catabolism enzymes, and/or bile salt hydrolases are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodimetns, the therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter E. coli_01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest.

In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-1, GLP-1 peptides, GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, SCFA producing enzymes, tryptophan catabolism enzymes, and/or bile salt hydrolases are secreted via Type I Hemolysin Secretion. In one embodiment, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of E. coli CFT073.

Essential Genes and Auxotrophs

As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, e.g., Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

An “essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the genetically engineered bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria.

Table 28 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

TABLE 28
Non-limiting Examples of Bacterial Genes Useful for Generation of an
Auxotroph
	Amino Acid	Oligonucleotide	Cell Wall
	cysE	thyA	dapA
	glnA	uraA	dapB
	ilvD		dapD
	leuB		dapE
	lysA		dapF
	serA
	metA
	glyA
	hisB
	ilvA
	pheA
	proA
	thrC
	trpC
	tyrA

Table 29 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

TABLE 29
Survival of amino acid auxotrophs in the mouse gut
Gene	AA Auxotroph	Pre-Gavage	24 hours	48 hours
argA	Arginine	Present	Present	Absent
cysE	Cysteine	Present	Present	Absent
glnA	Glutamine	Present	Present	Absent
glyA	Glycine	Present	Present	Absent
hisB	Histidine	Present	Present	Present
ilvA	Isoleucine	Present	Present	Absent
leuB	Leucine	Present	Present	Absent
lysA	Lysine	Present	Present	Absent
metA	Methionine	Present	Present	Present
pheA	Phenylalanine	Present	Present	Present
proA	Proline	Present	Present	Absent
serA	Serine	Present	Present	Present
thrC	Threonine	Present	Present	Present
trpC	Tryptophan	Present	Present	Present
tyrA	Tyrosine	Present	Present	Present
ilvD	Valine/Isoleucine/	Present	Present	Absent
	Leucine
thyA	Thiamine	Present	Absent	Absent
uraA	Uracil	Present	Absent	Absent
flhD	FlhD	Present	Present	Present

For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, 1pxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, mc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rp1J, rp1L, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, 1spA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, 1pxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rp1D, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rp10, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rp1X, rp1N, rpsQ, rpmC, rp1P, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, 1pxD, fabZ, 1pxA, 1pxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, 1pxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ, fabD, fabG, acpP, tmk, holB, 1o1C, 1o1D, 1o1E, purB, ymfK, minE, mind, pth, rsA, ispE, 1o1B, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, ”ACS Synthetic Biology (2015) DOI: 10.102¹/_acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G.

In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).

In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system shown in FIG. 68.

In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the genetically engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et al., 2015). In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the metabolic or satiety effector and/or immune modulator molecule.

Genetic Regulatory Circuits

In some embodiments, the genetically engineered bacteria comprise multi-layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811, incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that produce a metabolic or satiety effector and/or immune modulator molecule or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the metabolic or satiety effector and/or immune modulator molecule is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, LacI, CscR, DeoR, DgoR, FruR, GaIR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the metabolic or satiety effector and/or immune modulator molecule. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the metabolic or satiety effector and/or immune modulator molecule from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the gene or gene cassette remains in the 3′ to 5′ orientation, and no functional metabolic or satiety effector and/or immune modulator molecule is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the gene or gene cassette is reverted to the 5′ to 3′ orientation, and functional metabolic or satiety effector and/or immune modulator molecule is produced.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the metabolic or satiety effector and/or immune modulator molecule. The third gene encoding the T7 polymerase is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3′ to 5′ orientation, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5′ to 3′ orientation, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

Host-Plasmid Mutual Dependency

In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad-spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.

The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a metabolic or satiety effector and/or immune modulator molecule and further comprise a toxin-antitoxin system that simultaneously produces a toxin (hok) and a short-lived antitoxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp? from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).

Kill Switch

In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329, incorporated herein by reference in their entireties). The kill switch is intended to actively kill genetically engineered bacteria in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a metabolic or satiety effector and/or immune modulator molecule, or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of the metabolic or satiety effector and/or immune modulator molecule, e.g., GLP-1. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the metabolic or satiety effector and/or immune modulator molecule. Alternatively, the bacteria may be engineered to die after the bacterium has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl P-D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010).

Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low-oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.

In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxb1, PhiC31, TP901, Bxb1, PhiC31, TP901, HK022, HP1, R4, Intl, Int2, Int3, Int4, Int5, Int6, Intl, Int8, Int9, Int 10, Int 11, Int12, Int13, Int14, Int15, Int16, Int17, Int 18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.

In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in FIG. 69-FIG. 72. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the AraBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxing gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arbinoase system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an antitoxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

Arabinose inducible promoters are known in the art, including P_ara, P_araB, P_araC, and P_araBAD. In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the P_araC promoter and the P_araBAD promoter operate as a bidirectional promoter, with the P_araBAD promoter controlling expression of a heterologous gene(s) in one direction, and the P_araC (in close proximity to, and on the opposite strand from the P_araBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

In one exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P_araBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P_araC promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P_TetR). In the presence of arabinose, the AraC transcription factor activates the P_araBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the P_araBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the AraC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

In one embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the antitoxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

In another embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of the P_araBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

In another exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a _ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a P_arac promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the P_araBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the P_araBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of P_araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of P_araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin kill-switch system described directly above.

In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.

In some embodiments, the engineered bacteria of the present disclosure further comprise the gene(s) encoding the components of any of the above-described kill-switch circuits.

In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-051, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6, colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdID, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBl, hipB, MccE, MccE^cTD, MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

In some embodiments, the genetically engineered bacterium provided herein is an auxotroph. In one embodiment, the genetically engineered bacterium is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a ΔthyA and ΔdapA auxotroph.

In some embodiments, the genetically engineered bacterium provided herein further comprises a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

In some embodiments, the genetically engineered bacterium is an auxotroph comprising a therapeutic payload and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.

In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette for producing the metabolic or satiety effector and/or immune modulator molecule is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette for producing the metabolic or satiety effector and/or immune modulator molecule is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

Pharmaceutical Compositions and Formulations

Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a metabolic disease, e.g., obesity, type 2 diabetes. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers are provided.

In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria described herein that are engineered to treat, manage, ameliorate, and/or prevent a metabolic disease. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria described herein that are each engineered to treat, manage, ameliorate, and/or prevent a metabolic disease.

The pharmaceutical compositions of the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

The genetically engineered bacteria of the invention may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10⁵ to 10¹² bacteria, e.g., approximately 10⁵ bacteria, approximately 10⁶ bacteria, approximately 10⁷ bacteria, approximately 10⁸ bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents.

The genetically engineered bacteria of the invention may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

The genetically engineered bacteria of the invention may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulo se, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

In some embodiments, enteric coating materials may be used, in one or more coating layers (e.g., outer, inner and/o intermediate coating layers). Enteric coated polymers remain unionised at low pH, and therefore remain insoluble. But as the pH increases in the gastrointestinal tract, the acidic functional groups are capable of ionisation, and the polymer swells or becomes soluble in the intestinal fluid.

Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylo se acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.

Coating polymers also may comprise one or more of, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, EudragitTM S (poly(methacrylic acid, methyl methacrylate)1:2); Eudragit L100TM S (poly(methacrylic acid, methyl methacrylate)1:1); Eudragit L3ODTM, (poly(methacrylic acid, ethyl acrylate)1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate)1:1) (Eudragit™ L is an anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginate, vinyl acetate copolymers, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) (“Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof.

Coating layers may also include polymers which contain Hydroxypropylmethylcellulo se (HPMC), Hydroxypropylethylcellulose (HPEC), Hydroxypropylcellulo se (HPC), hydroxypropylethylcellulose (HPEC), hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydroxymethylethylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), methylhydroxyethylcellulose (M H EC), hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl hydroxyethylcellulose (CMHEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (HPCP), hydroxypropylethylcellulo se phthalate (HPECP), hydroxyproplymethylcellulose phthalate (HPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS).

Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria of the invention.

In one embodiment, the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, “flavor” is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

In certain embodiments, the genetically engineered bacteria of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

The genetically engineered bacteria of the invention may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The genetically engineered bacteria of the invention may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

In some embodiments, the invention provides pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

Single dosage forms of the pharmaceutical composition of the invention may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

Dosage regimens may be adjusted to provide a therapeutic response. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician.

In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions of the invention may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C. and 8° C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD₅₀, ED₅₀, EC₅₀, and IC₅₀ may be determined, and the dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

Methods of Treatment

Another aspect of the invention provides methods of treating metabolic disease, e.g., obesity, type 2 diabetes. In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility. In some embodiments, the subject to be treated is a human patient.

The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically.

In certain embodiments, the pharmaceutical composition described herein is administered to treat, manage, ameliorate, or prevent metabolic disease in a subject. In some embodiments, the method of treating or ameliorating metabolic disease allows one or more symptoms of the disease to improve by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the symptom (e.g., obesity, insulin resistance) is measured by comparing measurements in a subject before and after administration of the pharmaceutical composition.

Before, during, and after the administration of the pharmaceutical composition in a subject, metabolic symptoms and manifestations may be measured in a biological sample, e.g., blood, serum, plasma, urine, fecal matter, peritoneal fluid, a sample collected from a tissue, such as liver, skeletal muscle, pancreas, epididymal fat, subcutaneous fat, and beige fat. The biological samples may be analyzed to measure symptoms and manifestations of metabolic disease. Useful measurements include measures of lean mass, fat mass, body weight, food intake, GLP-1 levels, endotoxin levels, insulin levels, lipid levels, HbA1c levels, short-chain fatty acid levels, triglyceride levels, and nonesterified fatty acid levels. Useful assays include, but are not limited to, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests, assays for intestinal permeability, and assays for glycaemia upon multiple fasting and refeeding time points. In some embodiments, the methods may include administration of the compositions of the invention to reduce metabolic symptoms and manifestations to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions of the invention to reduce metabolic symptoms and manifestations to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's levels prior to treatment.

In certain embodiments, the genetically engineered bacteria are E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the genetically engineered bacteria may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in FIG. 84 and FIG. 85. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, e.g., insulin. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

Treatment In Vivo

The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a metabolic disease may be used (see, e.g., Mizoguchi 2012). In some embodiments, the animal is a C57BL/6J mouse that is fed a high fat diet in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia. In alternate embodiments, an animal harboring a genetic deficiency that causes a metabolic disease, e.g., a B6.BKS(D)-Lepr^db/db mouse, is used.

The genetically engineered bacteria of the invention are administered to the mice before, during, or after the onset of obesity and disease. Body weight, food intake, and blood plasma (e.g., triglyceride levels, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests) may be assayed to determine the severity and amelioration of disease. Metabolism and physical activity may be measured in metabolic cages. Animals may be sacrificed to assay metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology and gene expression.

TABLE 30
Summary of rodent models of type 2 diabetes
Induction
mechanism	Model	Main features	Possible uses
Obese models	Lepob/ob mice	Obesity-induced	Treatments to improve
(monogenic)		hyperglycaemia	insulin resistance
	Leprdb/db mice		Treatments to improve
	ZDF Rats		beta cell function
Obese models	KK mice	Obesity-induced	Treatments to improve
(polygenic)		hyperglycaemia	insulin resistance
	OLETF rat		Treatments to improve
			beta cell function
	NZO mice		Some models show
	TallyHo/Jng mice		diabetic complications
	NoncNZO10/LtJ mice
Induced obesity	High fat feeding (mice	Obesity-induced	Treatments to improve
	or rats)	hyperglycaemia	insulin resistance
	Desert gerbil		Treatments to improve
			beta cell function
	Nile grass rat		Treatments to prevent
			diet-induced obesity
Non-obese	GK rat	Hyperglycaemia	Treatments to improve
models		induced by	beta cell function
		insufficient beta	Treatments to improve
		cell function/mass	beta cell survival
Genetically	hIAPP mice	Amyloid	Treatments to prevent
induced models		deposition in islets	amyloid deposition
of beta cell			Treatments to improve
dysfunction			beta cell survival
	AKITA mice	Beta cell	Treatments to prevent ER
		destruction due to	stress
		ER stress.	Treatments to improve
			beta cell survival

As described in Aileen JF King, The use of animal models in diabetes research, Br J Pharmacol. 2012 June; 166(3): 877-894.

The engineered bacteria may be evaluated in vivo, e.g., in an animal model for NASH. Any suitable animal model of a disease associated with Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis (NAFLD/NASH) may be used. For example, the effects of liver steatosis and hepatic inflammation in an in vivo mouse model have been described (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice can be fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed.

An in vivo rat model of choline deficient/L-amino acid defined (CDAA) diet has also been described (Endo, et al., PLoS One, 8(5):e63388 (2013)). In this model, rats are fed the CDAA diet for eight weeks and then treated with a strain of Clostridium butyricum (MIYAIRI 588) two weeks after. The diet induces NAFLD/NASH symptoms such as liver steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocarcinogenesis. The rats are killed at 8, 16, and 50 weeks after completion of the diet regiments, and liver tissues removed and assayed.

Other models are known in the art, including a Lepob/Lepob and C57BL6 (B6) mouse model used to study the effects of high fat diet and GLP-1 administration within the NASH setting. See, for example, Trevaskis et al., Am. J. Physiology-Gastrointestinal and Liver Physiology, 302(8):G762-G772, 2012, and Takahashi et al., World J. Gastroenterol., 18(19):2300-2308, 2012, the entire contents of each of which are expressly incorporated herein by reference.

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EXAMPLES

The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

Example 1

Construction of Vectors for Producing Propionate

To facilitate inducible production of propionate in Escherichia coli Nissle, a propionate gene cassette comprising the genes encoding the enzymes of the acrylate pathway, i.e., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The genes are codon-optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). A second clone is generated as described above using a propionate gene cassette comprising the genes encoding the enzymes of the pyruvate pathway, i.e., thrA^fr, thrB, thrC, ilvA^fbr, aceE, aceF, and lpd; NCBI; Tseng et al., 2012). A third clone is generated as described above that comprises thrA^fbr, thrB, thrC, ilvA^fbr, aceE, aceF, lpd, and E. coli tesB. Each propionate gene cassette is expressed under the control of each of the following regulatory regions: a FNR-inducible regulatory region selected from the sequences listed in Table 21, a tetracycline-inducible promoter, and an arabinose-inducible promoter. In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of propionate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.

The propionate construct is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., FIG. 57). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate.

Example 2

Lambda Red Recombination

Lambda red recombination is used to make chromosomal modifications, e.g., to express a propionate biosynthesis cassette in E. coli Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli . A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD₆₀₀ of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C. for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.

DNA sequences comprising the desired propionate biosynthesis genes shown above were ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. In some embodiments, the construct is in the E. coli Nissle genome at the malP/T site (FIG. 57). To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting construct comprises approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the propionate biosynthesis genes, and approximately 50 bases of homology downstream.

Example 3

Transforming E. coli

Each of the constructs above is transformed into E. coli Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1:100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD₆₀₀ of 0.1. 0.05 mL of 100× L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD₆₀₀ of 0.4-0.6. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of the construct is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing kanamycin and incubated overnight.

In alternate embodiments, the propionate cassette may be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are shown in FIGS. 1-5. To create a vector capable of integrating the synthesized propionate cassette construct into the chromosome, Gibson assembly was first used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the propionate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20 ug/mL at 37 degrees C. Growth at 37 degrees C. also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac−).

Example 4

Verifying Mutants

The presence of the propionate gene cassette is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 pi of cold ddH₂O by pipetting up and down. 3 μl of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C. overnight. A PCR master mix is made using 5 μl of 10× PCR buffer, 0.6 μl of 10 mM dNTPs, 0.4 μl of 50 mM Mg₂SO₄, 6.0 μl of 10× enhancer, and 3.0 μl of ddH₂O (15 μl of master mix per PCR reaction). A 10 μM primer mix is made by mixing 2 μL of primers unique to the propionate construct (100 μM stock) into 16 μL of ddH₂O. For each 20 μl reaction, 15 μL of the PCR master mix, 2.0 μL of the colony suspension (template), 2.0 μL of the primer mix, and 1.0 μL of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C. at 5:00 min., 2) 94° C. at 0:15 min., 3) 55° C. at 0:30 min., 4) 68° C. at 2:00 min., 5) 68° C. at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μL of each amplicon and 2.5 μL 5× dye. The PCR product only forms if the heterologous sequence has been inserted.

Example 5

Generation of ΔThyA

An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a 100 um concentration are found in Table 31.

TABLE 31
Primer Sequences
			SEQ ID
Name	Sequence	Description	NO
SR36	tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAG	Round 1: binds	SEQ ID
	GCTGGAGCTGCTTC	on pKD3	NO: 215
SR38	gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAA	Round 1: binds	SEQ ID
	TATCCTCCTTAG	on pKD3	NO: 216
SR33	caacacgtttcctgaggaaccatgaaacagtatttagaactgatgcaaaaag	Round 2: binds	SEQ ID
		to round 1 PCR	NO: 217
		product
SR34	cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc	Round 2: binds	SEQ ID
		to round 1 PCR	NO: 218
		product
SR43	atatcgtcgcagcccacagcaacacgtttcctgagg	Round 3: binds	SEQ ID
		to round 2 PCR	NO: 219
		product
SR44	aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc	Round 3: binds	SEQ ID
		to round 2 PCR	NO: 220
		product

For the first PCR round, 4×50 ul PCR reactions containing ing pKD3 as template, 25 ul 2×phusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6 ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:

stepl: 98 c for 30 s

step2: 98 c for 10 s

step3: 55 c for 15 s

step4: 72 c for 20 s

repeat step 2-4 for 30 cycles

step5: 72 c for 5 min

Subsequently, 5 ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30 ul nuclease free water.

For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4×50 ul PCR reactions as described above except with 0.2 ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30 ul as described above.

For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4×50 ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, lml SOC medium containing 3 mM thymidine was added, and cells were allowed to recover at 37 C for 2 h with shaking. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 20 ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. +cam 20 ug/ml + or − thy 3 mM. (thyA auxotrophs will only grow in media supplemented with thy 3 mM).

Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37° C. until OD600=0.4-0.6. 1mL of cells were washed as follows: cells were pelleted at 16,000×g for 1 minute. The supernatant was discarded and the pellet was resuspended in 1 mL ice-cold 10% glycerol. This wash step was repeated 3× times. The final pellet was resuspended in 70 ul ice-cold 10% glycerol. Next, cells were electroporated with ing pCP20 plasmid DNA, and 1 mL SOC supplemented with 3 mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30° C. for lhours. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 100ug/mlcarbenicillin and grown at 30° C. for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42° C.

To test the colony-purified transformants, a colony was picked from the 42° C. plate with a pipette tip and resuspended in 10 μL LB. 3 μl, of the cell suspension was pipetted onto a set of 3 plates: Cam, (37° C.; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30° C., tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37° C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37° C.

Example 6

Production of Propionate in Genetically Engineered E. coli

Production of propionate is assessed in E. coli Nissle strains containing the propionate cassettes described above. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the propionate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h, and the inducible constructs are induced as follows: (1) bacteria comprising a propionate gene cassette driven by a FNR-inducible promoter are induced in LB at 37C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2, and 20 mM nitrate) at 37° C.; (2) bacteria comprising a propionate gene cassette driven by a tetracycline-inducible promoter are induced with anhydrotetracycline (100 ng/mL); (3) bacteria comprising a propionate gene cassette driven by a arabinose-inducible promoter are induced with 1% arabinose in media lacking glucose. One mL culture aliquots are prepared in 1.5 mL capped tubes and FNR-inducible constructs are incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for propionate concentration by LC-MS to confirm that propionate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 7

Efficacy of Propionate-Expressing Bacteria in a Mouse Model of Obesity and Type 2 Diabetes Mellitus (T2DM)

For in vivo studies to assess the efficacy of the genetically engineered bacteria in an animal model of obesity and type 2 diabetes, C57BL/6J mice are fed a high fat diet (60 kcal % fat, Research Diets Inc.) starting from 4-5 weeks of age for 8 weeks or until body weight is at least 45 g in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia, e.g., glycaemia above 160 mg/dL and plasma insulin above 4000 pg/mL. Alternatively, B6.BKS(D)-Lepr^db/db mice (Lepr^db/db) are obtained from The Jackson Laboratory; these mice typically become obese and display T2DM-related symptoms beginning at 10 weeks of age.

Bacteria harboring the propionate gene cassette described above are grown overnight in LB. Bacteria are then diluted 1:100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. To analyze the efficacy of the bacteria in vivo, bacteria are resuspended in phosphate buffered saline (PBS) and 100 microliters is administered by oral gavage to mice daily for 8 weeks. Alternatively, the bacteria can be supplemented in the drinking water (5×10⁹ CFU bacteria/mL).

Body weight and food intake are measured weekly before, during, and after the administration of the bacteria. In addition, mice are subjected to insulin tolerance tests (ITT), glucose tolerance tests (GTT) and pyruvate tolerance tests (PTT) to determine the severity of T2DM during treatment, e.g., amelioration of insulin resistance. For ITT, mice are fasted overnight and injected with insulin (1 U/kg, diluted in PBS). Blood glucose levels are measured prior to the injection and at 20, 40, 60, and 90 min. post injection via tail bleeding. For GTT, mice are fasted overnight and injected with glucose solution (1 g/kg, dissolved in PBS); blood glucose levels are measured as described above in order to determine changes. For PTT, mice are fasted overnight and injected with sodium pyruvate solution (lg/kg, dissolved in PBS); blood glucose levels are measured as described above. Whole-body metabolic functions are analyzed by placing the mice in a Comprehensive Lab Animal Monitoring System (CLAMS), which monitors physical activity, food intake, metabolic rate (as a function of O₂ consumption and CO₂ production). Mice are sacrificed and metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology, e.g., Oil Red O staining of the liver, and gene expression.

Example 8

Nissle Residence

Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non-limiting example using a streptomycin-resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 32. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted.

TABLE 32
CFU administered via oral gavage
CFU administered via oral gavage
	Strain	Day 1	Day 2	Day 3
	SYN-103	1.30E+08	8.50E+08	1.90E+09

On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1-6; Table 14). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted.

Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 33.

FIG. 84 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

TABLE 33
Nissle residence in vivo
ID	Day 2	Day 3	Day 4	Day 5
1	2.40E+05	6.50E+03	6.00E+04	2.00E+03
2	1.00E+05	1.00E+04	3.30E+04	3.00E+03
3	6.00E+04	1.70E+04	6.30E+04	2.00E+02
4	3.00E+04	1.50E+04	1.10E+05	3.00E+02
5		1.00E+04	3.00E+05	1.50E+04
6		1.00E+06	4.00E+05	2.30E+04
Avg	1.08E+05	1.76E+05	1.61E+05	7.25E+03
ID	Day 6	Day 7	Day 8	Day 9	Day 10
1	9.10E+03	1.70E+03	4.30E+03	6.40E+03	2.77E+03
2	6.00E+03	7.00E+02	6.00E+02	0.00E+00	0.00E+00
3	1.00E+02	2.00E+02	0.00E+00	0.00E+00	0.00E+00
4	1.50E+03	1.00E+02		0.00E+00	0.00E+00
5	3.10E+04	3.60E+03		0.00E+00	0.00E+00
6	1.50E+03	1.40E+03	4.20E+03	1.00E+02	0.00E+00
Avg	8.20E+03	1.28E+03	2.28E+03	1.08E+03	4.62E+02

Example 9

Intestinal Residence and Survival of Bacterial Strains In Vivo

Localization and intestinal residence time of streptomycin resistant Nissle, FIG. 85) was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10¹⁰ CFU/mL. Mice (C57BL6/J, 10-12 weeks old) were gavaged with 100 μL of bacteria (approximately 10⁹ CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37° C. overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in FIG. 85.

Example 10

Construction of Vectors for Overproducing Butyrate

In addition to the ammonia conversion circuit, GABA transport circuit, GABA metabolic circuit, and/or manganese transport circuit described above, the E. coli Nissle bacteria further comprise one or more circuits for producing a gut barrier enhancer molecule.

To facilitate inducible production of butyrate in E. coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The butyrate gene cassette is placed under the control of a FNR regulatory region selected from (SEQ ID NOs: 177-188) (Table 18 or Table 19) In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Tables 34 and 35). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Tables 14-17). In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.

TABLE 34
pLogic031-nsrR-norB-butyrate construct (SEQ ID NO: 221)
                              Nucleotide sequences of pLogic031-nsrR-norB-butyrate
Description                   construct (SEQ ID NO: 221)
Nucleic acid                  ttattatcgcaccgcaatcgggattttcgattcataaagcagg
sequence of an                tcgtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgt
exemplary RNS-                ccagatacgtgaaaaacgacttcattgcaccgccgagtatgcc
regulated                     cgtcagccggcaggacggcgtaatcaggcattcgttgttcggg
construct                     cccatacactcgaccagctgcatcggttcgaggtggcggacga
comprising a gene             ccgcgccgatattgatgcgttcgggcggcgcggccagcctcag
encoding nsrR, a              cccgccgcctttcccgcgtacgctgtgcaagaacccgcctttg
regulatory region             accagcgcggtaaccactttcatcaaatggcttttggaaatgc
of norB, and a                cgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtc
butyrogenic gene              gttgacggcggtgtagatgaggacgcgcagcccgtagtcggta
cassette                      tgttgggtcagatacat              acaacctccttagtacatgcaaaatt
(pLogic031-nsrR-              atttctagagcaacatacgagccggaagcataaagtgtaaagc
norB-butyrate                 ctggggtgcctaatgagttgagttgaggaattataacaggaag
construct; SEQ. ID            aaatattcctcatacgcttgtaattcctctatggttgttgaca
NO: 79). The
sequence
encoding NsrR is              gatatacatatggatttaaattctaaaaaatatcagatgctta
underlined and                aagagctatatgtaagcttcgctgaaaatgaagttaaaccttt
bolded              , and the agcaacagaacttgatgaagaagaaagatttccttatgaaaca
NsrR binding site,            gtggaaaaaatggcaaaagcaggaatgatgggtataccatatc
i.e., a regulatory            caaaagaatatggtggagaaggtggagacactgtaggatatat
region of norB is             aatggcagttgaagaattgtctagagtttgtggtactacagga
                              gttatattatcagctcatacatctcttggctcatggcctatat
                              atcaatatggtaatgaagaacaaaaacaaaaattcttaagacc
                              actagcaagtggagaaaaattaggagcatttggtcttactgag
                              cctaatgctggtacagatgcgtctggccaacaaacaactgctg
                              ttttagacggggatgaatacatacttaatggctcaaaaatatt
                              tataacaaacgcaatagctggtgacatatatgtagtaatggca
                              atgactgataaatctaaggggaacaaaggaatatcagcattta
                              tagttgaaaaaggaactcctgggtttagctttggagttaaaga
                              aaagaaaatgggtataagaggttcagctacgagtgaattaata
                              tttgaggattgcagaatacctaaagaaaatttacttggaaaag
                              aaggtcaaggatttaagatagcaatgtctactcttgatggtgg
                              tagaattggtatagctgcacaagctttaggtttagcacaaggt
                              gctcttgatgaaactgttaaatatgtaaaagaaagagtacaat
                              ttggtagaccattatcaaaattccaaaatacacaattccaatt
                              agctgatatggaagttaaggtacaagcggctagacaccttgta
                              tatcaagcagctataaataaagacttaggaaaaccttatggag
                              tagaagcagcaatggcaaaattatttgcagctgaaacagctat
                              ggaagttactacaaaagctgtacaacttcatggaggatatgga
                              tacactcgtgactatccagtagaaagaatgatgagagatgcta
                              agataactgaaatatatgaaggaactagtgaagttcaaagaat
                              ggttatttcaggaaaactattaaaatagtaagaaggagatata
                              catatggaggaaggatttatgaatatagtcgtttgtataaaac
                              aagttccagatacaacagaagttaaactagatcctaatacagg
                              tactttaattagagatggagtaccaagtataataaaccctgat
                              gataaagcaggtttagaagaagctataaaattaaaagaagaaa
                              tgggtgctcatgtaactgttataacaatgggacctcctcaagc
                              agatatggctttaaaagaagctttagcaatgggtgcagataga
                              ggtatattattaacagatagagcatttgcgggtgctgatactt
                              gggcaacttcatcagcattagcaggagcattaaaaaatataga
                              ttttgatattataatagctggaagacaggcgatagatggagat
                              actgcacaagttggacctcaaatagctgaacatttaaatcttc
                              catcaataacatatgctgaagaaataaaaactgaaggtgaata
                              tgtattagtaaaaagacaatttgaagattgttgccatgactta
                              aaagttaaaatgccatgccttataacaactcttaaagatatga
                              acacaccaagatacatgaaagttggaagaatatatgatgcttt
                              cgaaaatgatgtagtagaaacatggactgtaaaagatatagaa
                              gttgacccttctaatttaggtcttaaaggttctccaactagtg
                              tatttaaatcatttacaaaatcagttaaaccagctggtacaat
                              atacaatgaagatgcgaaaacatcagctggaattatcatagat
                              aaattaaaagagaagtatatcatataataagaaggagatatac
                              atatgggtaacgttttagtagtaatagaacaaagagaaaatgt
                              aattcaaactgtttctttagaattactaggaaaggctacagaa
                              atagcaaaagattatgatacaaaagtttctgcattacttttag
                              gtagtaaggtagaaggtttaatagatacattagcacactatgg
                              tgcagatgaggtaatagtagtagatgatgaagctttagcagtg
                              tatacaactgaaccatatacaaaagcagcttatgaagcaataa
                              aagcagctgaccctatagttgtattatttggtgcaacttcaat
                              aggtagagatttagcgcctagagtttctgctagaatacataca
                              ggtcttactgctgactgtacaggtcttgcagtagctgaagata
                              caaaattattattaatgacaagacctgcctttggtggaaatat
                              aatggcaacaatagtttgtaaagatttcagacctcaaatgtct
                              acagttagaccaggggttatgaagaaaaatgaacctgatgaaa
                              ctaaagaagctgtaattaaccgtttcaaggtagaatttaatga
                              tgctgataaattagttcaagttgtacaagtaataaaagaagct
                              aaaaaacaagttaaaatagaagatgctaagatattagtttctg
                              ctggacgtggaatgggtggaaaagaaaacttagacatacttta
                              tgaattagctgaaattataggtggagaagtttctggttctcgt
                              gccactatagatgcaggttggttagataaagcaagacaagttg
                              gtcaaactggtaaaactgtaagaccagacctttatatagcatg
                              tggtatatctggagcaatacaacatatagctggtatggaagat
                              gctgagtttatagttgctataaataaaaatccagaagctccaa
                              tatttaaatatgctgatgttggtatagttggagatgttcataa
                              agtgcttccagaacttatcagtcagttaagtgttgcaaaagaa
                              aaaggtgaagttttagctaactaataagaaggagatatacata
                              tgagagaagtagtaattgccagtgcagctagaacagcagtagg
                              aagttttggaggagcatttaaatcagtttcagcggtagagtta
                              ggggtaacagcagctaaagaagctataaaaagagctaacataa
                              ctccagatatgatagatgaatctcttttagggggagtacttac
                              agcaggtcttggacaaaatatagcaagacaaatagcattagga
                              gcaggaataccagtagaaaaaccagctatgactataaatatag
                              tttgtggttctggattaagatctgtttcaatggcatctcaact
                              tatagcattaggtgatgctgatataatgttagttggtggagct
                              gaaaacatgagtatgtctccttatttagtaccaagtgcgagat
                              atggtgcaagaatgggtgatgctgcttttgttgattcaatgat
                              aaaagatggattatcagacatatttaataactatcacatgggt
                              attactgctgaaaacatagcagagcaatggaatataactagag
                              aagaacaagatgaattagctcttgcaagtcaaaataaagctga
                              aaaagctcaagctgaaggaaaatttgatgaagaaatagttcct
                              gttgttataaaaggaagaaaaggtgacactgtagtagataaag
                              atgaatatattaagcctggcactacaatggagaaacttgctaa
                              gttaagacctgcatttaaaaaagatggaacagttactgctggt
                              aatgcatcaggaataaatgatggtgctgctatgttagtagtaa
                              tggctaaagaaaaagctgaagaactaggaatagagcctcttgc
                              aactatagtttcttatggaacagctggtgttgaccctaaaata
                              atgggatatggaccagttccagcaactaaaaaagctttagaag
                              ctgctaatatgactattgaagatatagatttagttgaagctaa
                              tgaggcatttgctgcccaatctgtagctgtaataagagactta
                              aatatagatatgaataaagttaatgttaatggtggagcaatag
                              ctataggacatccaataggatgctcaggagcaagaatacttac
                              tacacttttatatgaaatgaagagaagagatgctaaaactggt
                              cttgctacactttgtataggcggtggaatgggaactactttaa
                              tagttaagagatagtaagaaggagatatacatatgaaattagc
                              tgtaataggtagtggaactatgggaagtggtattgtacaaact
                              tttgcaagttgtggacatgatgtatgtttaaagagtagaactc
                              aaggtgctatagataaatgtttagctttattagataaaaattt
                              aactaagttagttactaagggaaaaatggatgaagctacaaaa
                              gcagaaatattaagtcatgttagttcaactactaattatgaag
                              atttaaaagatatggatttaataatagaagcatctgtagaaga
                              catgaatataaagaaagatgttttcaagttactagatgaatta
                              tgtaaagaagatactatcttggcaacaaatacttcatcattat
                              ctataacagaaatagcttcttctactaagcgcccagataaagt
                              tataggaatgcatttctttaatccagttcctatgatgaaatta
                              gttgaagttataagtggtcagttaacatcaaaagttacttttg
                              atacagtatttgaattatctaagagtatcaataaagtaccagt
                              agatgtatctgaatctcctggatttgtagtaaatagaatactt
                              atacctatgataaatgaagctgttggtatatatgcagatggtg
                              ttgcaagtaaagaagaaatagatgaagctatgaaattaggagc
                              aaaccatccaatgggaccactagcattaggtgatttaatcgga
                              ttagatgttgttttagctataatgaacgttttatatactgaat
                              ttggagatactaaatatagacctcatccacttttagctaaaat
                              ggttagagctaatcaattaggaagaaaaactaagataggattc
                              tatgattataataaataataagaaggagatatacatatgagta
                              caagtgatgttaaagtttatgagaatgtagctgttgaagtaga
                              tggaaatatatgtacagtgaaaatgaatagacctaaagccctt
                              aatgcaataaattcaaagactttagaagaactttatgaagtat
                              ttgtagatattaataatgatgaaactattgatgttgtaatatt
                              gacaggggaaggaaaggcatttgtagctggagcagatattgca
                              tacatgaaagatttagatgctgtagctgctaaagattttagta
                              tcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa
                              agtagtgatagctgctgtaaacggatttgctttaggtggagga
                              tgtgaacttgcaatggcatgtgatataagaattgcatctgcta
                              aagctaaatttggtcagccagaagtaactcttggaataactcc
                              aggatatggaggaactcaaaggcttacaagattggttggaatg
                              gcaaaagcaaaagaattaatctttacaggtcaagttataaaag
                              ctgatgaagctgaaaaaatagggctagtaaatagagtcgttga
                              gccagacattttaatagaagaagttgagaaattagctaagata
                              atagctaaaaatgctcagcttgcagttagatactctaaagaag
                              caatacaacttggtgctcaaactgatataaatactggaataga
                              tatagaatctaatttatttggtctttgtttttcaactaaagac
                              caaaaagaaggaatgtcagctttcgttgaaaagagagaagcta
                              actttataaaagggtaataagaaggagatatacatatgagaag
                              ttttgaagaagtaattaagtttgcaaaagaaagaggacctaaa
                              actatatcagtagcatgttgccaagataaagaagttttaatgg
                              cagttgaaatggctagaaaagaaaaaatagcaaatgccatttt
                              agtaggagatatagaaaagactaaagaaattgcaaaaagcata
                              gacatggatatcgaaaattatgaactgatagatataaaagatt
                              tagcagaagcatctctaaaatctgttgaattagtttcacaagg
                              aaaagccgacatggtaatgaaaggcttagtagacacatcaata
                              atactaaaagcagttttaaataaagaagtaggtcttagaactg
                              gaaatgtattaagtcacgtagcagtatttgatgtagagggata
                              tgatagattatttttcgtaactgacgcagctatgaacttagct
                              cctgatacaaatactaaaaagcaaatcatagaaaatgcttgca
                              cagtagcacattcattagatataagtgaaccaaaagttgctgc
                              aatatgcgcaaaagaaaaagtaaatccaaaaatgaaagataca
                              gttgaagctaaagaactagaagaaatgtatgaaagaggagaaa
                              tcaaaggttgtatggttggtgggccttttgcaattgataatgc
                              agtatctttagaagcagctaaacataaaggtataaatcatcct
                              gtagcaggacgagctgatatattattagccccagatattgaag
                              gtggtaacatattatataaagctttggtattcttctcaaaatc
                              aaaaaatgcaggagttatagttggggctaaagcaccaataata
                              ttaacttctagagcagacagtgaagaaactaaactaaactcaa
                              tagctttaggtgttttaatggcagcaaaggcataataagaagg
                              agatatacatatgagcaaaatatttaaaatcttaacaataaat
                              cctggttcgacatcaactaaaatagctgtatttgataatgagg
                              atttagtatttgaaaaaactttaagacattcttcagaagaaat
                              aggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaa
                              caagtaatagaagaagctctaaaagaaggtggagtaaaaacat
                              ctgaattagatgctgtagtaggtagaggaggacttcttaaacc
                              tataaaaggtggtacttattcagtaagtgctgctatgattgaa
                              gatttaaaagtgggagttttaggagaacacgcttcaaacctag
                              gtggaataatagcaaaacaaataggtgaagaagtaaatgttcc
                              ttcatacatagtagaccctgttgttgtagatgaattagaagat
                              gttgctagaatttctggtatgcctgaaataagtagagcaagtg
                              tagtacatgctttaaatcaaaaggcaatagcaagaagatatgc
                              tagagaaataaacaagaaatatgaagatataaatcttatagtt
                              gcacacatgggtggaggagtttctgttggagctcataaaaatg
                              gtaaaatagtagatgttgcaaacgcattagatggagaaggacc
                              tttctctccagaaagaagtggtggactaccagtaggtgcatta
                              gtaaaaatgtgctttagtggaaaatatactcaagatgaaatta
                              aaaagaaaataaaaggtaatggcggactagttgcatacttaaa
                              cactaatgatgctagagaagttgaagaaagaattgaagctggt
                              gatgaaaaagctaaattagtatatgaagctatggcatatcaaa
                              tctctaaagaaataggagctagtgctgcagttcttaagggaga
                              tgtaaaagcaatattattaactggtggaatcgcatattcaaaa
                              atgtttacagaaatgattgcagatagagttaaatttatagcag
                              atgtaaaagtttatccaggtgaagatgaaatgattgcattagc
                              tcaaggtggacttagagttttaactggtgaagaagaggctcaa
                              gtttatgataactaataa

TABLE 35
Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct
                Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct
Description     (SEQ ID NO: 222)
Nucleic acid    ttattatcgcaccgcaatcgggattttcgattcataaagcaggtc
sequence of an  gtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgtccag
exemplary RNS-  atacgtgaaaaacgacttcattgcaccgccgagtatgcccgtcag
regulated       ccggcaggacggcgtaatcaggcattcgttgttcgggcccataca
construct       ctcgaccagctgcatcggttcgaggtggcggacgaccgcgccgat
comprising a    attgatgcgttcgggcggcgcggccagcctcagcccgccgccttt
gene encoding   cccgcgtacgctgtgcaagaacccgcctttgaccagcgcggtaac
nsrR, a         cactttcatcaaatggcttttggaaatgccgtaggtcgaggcgat
regulatory      ggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagat
region of norB, gaggacgcgcagcccgtagtcggtatgttgggtcagatacat              aca
and a           acctccttagtacatgcaaaattatttctagagcaacatacgagc
butyrogenic     cggaagcataaagtgtaaagcctggggtgcctaatgagttgagtt
gene cassette   gaggaattataacaggaagaaatattcctcatacgcttgtaattc
(pLogic046-
nsrR-norB-
butyrate        actttaagaaggagatatacatatgatcgtaaaacctatggtacg
construct; SEQ. caacaatatctgcctgaacgcccatcctcagggctgcaagaaggg
ID NO: 80).     agtggaagatcagattgaatataccaagaaacgcattaccgcaga
                agtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg
                ctgctcaaatggttacggcctggcgagccgcattactgctgcgtt
                cggatacggggctgcgaccatcggcgtgtcctttgaaaaagcggg
                ttcagaaaccaaatatggtacaccgggatggtacaataatttggc
                atttgatgaagcggcaaaacgcgagggtctttatagcgtgacgat
                cgacggcgatgcgttttcagacgagatcaaggcccaggtaattga
                ggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag
                cttggccagcccagtacgtactgatcctgatacaggtatcatgca
                caaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaac
                agtagatccgtttactggcgagctgaaggaaatctccgcggaacc
                agcaaatgacgaggaagcagccgccactgttaaagttatgggggg
                tgaagattgggaacgttggattaagcagctgtcgaaggaaggcct
                cttagaagaaggctgtattaccttggcctatagttatattggccc
                tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggc
                caaagaacacctggaggccacagcacaccgtctcaacaaagagaa
                cccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaac
                ccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag
                cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtat
                tgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaaga
                tggtacaattccagttgatgaggaaaatcgcattcgcattgatga
                ttgggagttagaagaagacgtccagaaagcggtatccgcgttgat
                ggagaaagtcacgggtgaaaacgcagaatctctcactgacttagc
                ggggtaccgccatgatttcttagctagtaacggctttgatgtaga
                aggtattaattatgaagcggaagttgaacgcttcgaccgtatctg
                ataagaaggagatatacatatgagagaagtagtaattgccagtgc
                agctagaacagcagtaggaagttttggaggagcatttaaatcagt
                ttcagcggtagagttaggggtaacagcagctaaagaagctataaa
                aagagctaacataactccagatatgatagatgaatctcttttagg
                gggagtacttacagcaggtcttggacaaaatatagcaagacaaat
                agcattaggagcaggaataccagtagaaaaaccagctatgactat
                aaatatagtttgtggttctggattaagatctgtttcaatggcatc
                tcaacttatagcattaggtgatgctgatataatgttagttggtgg
                agctgaaaacatgagtatgtctccttatttagtaccaagtgcgag
                atatggtgcaagaatgggtgatgctgcttttgttgattcaatgat
                aaaagatggattatcagacatatttaataactatcacatgggtat
                tactgctgaaaacatagcagagcaatggaatataactagagaaga
                acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagc
                tcaagctgaaggaaaatttgatgaagaaatagttcctgttgttat
                aaaaggaagaaaaggtgacactgtagtagataaagatgaatatat
                taagcctggcactacaatggagaaacttgctaagttaagacctgc
                atttaaaaaagatggaacagttactgctggtaatgcatcaggaat
                aaatgatggtgctgctatgttagtagtaatggctaaagaaaaagc
                tgaagaactaggaatagagcctcttgcaactatagtttcttatgg
                aacagctggtgttgaccctaaaataatgggatatggaccagttcc
                agcaactaaaaaagctttagaagctgctaatatgactattgaaga
                tatagatttagttgaagctaatgaggcatttgctgcccaatctgt
                agctgtaataagagacttaaatatagatatgaataaagttaatgt
                taatggtggagcaatagctataggacatccaataggatgctcagg
                agcaagaatacttactacacttttatatgaaatgaagagaagaga
                tgctaaaactggtcttgctacactttgtataggcggtggaatggg
                aactactttaatagttaagagatagtaagaaggagatatacatat
                gaaattagctgtaataggtagtggaactatgggaagtggtattgt
                acaaacttttgcaagttgtggacatgatgtatgtttaaagagtag
                aactcaaggtgctatagataaatgtttagctttattagataaaaa
                tttaactaagttagttactaagggaaaaatggatgaagctacaaa
                agcagaaatattaagtcatgttagttcaactactaattatgaaga
                tttaaaagatatggatttaataatagaagcatctgtagaagacat
                gaatataaagaaagatgttttcaagttactagatgaattatgtaa
                agaagatactatcttggcaacaaatacttcatcattatctataac
                agaaatagcttcttctactaagcgcccagataaagttataggaat
                gcatttctttaatccagttcctatgatgaaattagttgaagttat
                aagtggtcagttaacatcaaaagttacttttgatacagtatttga
                attatctaagagtatcaataaagtaccagtagatgtatctgaatc
                tcctggatttgtagtaaatagaatacttatacctatgataaatga
                agctgttggtatatatgcagatggtgttgcaagtaaagaagaaat
                agatgaagctatgaaattaggagcaaaccatccaatgggaccact
                agcattaggtgatttaatcggattagatgttgttttagctataat
                gaacgttttatatactgaatttggagatactaaatatagacctca
                tccacttttagctaaaatggttagagctaatcaattaggaagaaa
                aactaagataggattctatgattataataaataataagaaggaga
                tatacatatgagtacaagtgatgttaaagtttatgagaatgtagc
                tgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc
                taaagcccttaatgcaataaattcaaagactttagaagaacttta
                tgaagtatttgtagatattaataatgatgaaactattgatgttgt
                aatattgacaggggaaggaaaggcatttgtagctggagcagatat
                tgcatacatgaaagatttagatgctgtagctgctaaagattttag
                tatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa
                agtagtgatagctgctgtaaacggatttgctttaggtggaggatg
                tgaacttgcaatggcatgtgatataagaattgcatctgctaaagc
                taaatttggtcagccagaagtaactcttggaataactccaggata
                tggaggaactcaaaggcttacaagattggttggaatggcaaaagc
                aaaagaattaatctttacaggtcaagttataaaagctgatgaagc
                tgaaaaaatagggctagtaaatagagtcgttgagccagacatttt
                aatagaagaagttgagaaattagctaagataatagctaaaaatgc
                tcagcttgcagttagatactctaaagaagcaatacaacttggtgc
                tcaaactgatataaatactggaatagatatagaatctaatttatt
                tggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagc
                tttcgttgaaaagagagaagctaactttataaaagggtaataaga
                aggagatatacatatgagaagttttgaagaagtaattaagtttgc
                aaaagaaagaggacctaaaactatatcagtagcatgttgccaaga
                taaagaagttttaatggcagttgaaatggctagaaaagaaaaaat
                agcaaatgccattttagtaggagatatagaaaagactaaagaaat
                tgcaaaaagcatagacatggatatcgaaaattatgaactgataga
                tataaaagatttagcagaagcatctctaaaatctgttgaattagt
                ttcacaaggaaaagccgacatggtaatgaaaggcttagtagacac
                atcaataatactaaaagcagttttaaataaagaagtaggtcttag
                aactggaaatgtattaagtcacgtagcagtatttgatgtagaggg
                atatgatagattatttttcgtaactgacgcagctatgaacttagc
                tcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcac
                agtagcacattcattagatataagtgaaccaaaagttgctgcaat
                atgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttga
                agctaaagaactagaagaaatgtatgaaagaggagaaatcaaagg
                ttgtatggttggtgggccttttgcaattgataatgcagtatcttt
                agaagcagctaaacataaaggtataaatcatcctgtagcaggacg
                agctgatatattattagccccagatattgaaggtggtaacatatt
                atataaagctttggtattcttctcaaaatcaaaaaatgcaggagt
                tatagttggggctaaagcaccaataatattaacttctagagcaga
                cagtgaagaaactaaactaaactcaatagctttaggtgttttaat
                ggcagcaaaggcataataagaaggagatatacatatgagcaaaat
                atttaaaatcttaacaataaatcctggttcgacatcaactaaaat
                agctgtatttgataatgaggatttagtatttgaaaaaactttaag
                acattcttcagaagaaataggaaaatatgagaaggtgtctgacca
                atttgaatttcgtaaacaagtaatagaagaagctctaaaagaagg
                tggagtaaaaacatctgaattagatgctgtagtaggtagaggagg
                acttcttaaacctataaaaggtggtacttattcagtaagtgctgc
                tatgattgaagatttaaaagtgggagttttaggagaacacgcttc
                aaacctaggtggaataatagcaaaacaaataggtgaagaagtaaa
                tgttccttcatacatagtagaccctgttgttgtagatgaattaga
                agatgttgctagaatttctggtatgcctgaaataagtagagcaag
                tgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgc
                tagagaaataaacaagaaatatgaagatataaatcttatagttgc
                acacatgggtggaggagtttctgttggagctcataaaaatggtaa
                aatagtagatgttgcaaacgcattagatggagaaggacctttctc
                tccagaaagaagtggtggactaccagtaggtgcattagtaaaaat
                gtgctttagtggaaaatatactcaagatgaaattaaaaagaaaat
                aaaaggtaatggcggactagttgcatacttaaacactaatgatgc
                tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaa
                attagtatatgaagctatggcatatcaaatctctaaagaaatagg
                agctagtgctgcagttcttaagggagatgtaaaagcaatattatt
                aactggtggaatcgcatattcaaaaatgtttacagaaatgattgc
                agatagagttaaatttatagcagatgtaaaagtttatccaggtga
                agatgaaatgattgcattagctcaaggtggacttagagttttaac
                tggtgaagaagaggctcaagtttatgataactaataa

The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA and may exhibit dependence on oxygen as a co-oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, a second butyrate gene cassette in which the ter gene replaces the bcd2-etfA3-etfB3 genes of the first butyrate cassette is synthesized (Genewiz, Cambridge, Mass.). The ter gene is codon-optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). The second butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The second butyrate gene cassette is placed under control of a FNR regulatory region as described above. In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 20). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 21 and Table 22).

TABLE 36
ROS regulated constructs, OxyR construct, Tet-regulated constructs
Description      Sequence
Nucleotide       ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtagaatagcaatgaacgattat
sequences of     ccctatcaagcattctgactgataattgctcacacgaattcattaaagaggagaaaggtaccatggatttaa
pLogic031-       attctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaaatgaagttaaacctttagcaac
oxyS-butyrate    agaacttgatgaagaagaaagatttccttatgaaacagtggaaaaaatggcaaaagcaggaatgatggg
construct (SEQ   tataccatatccaaaagaatatggtggagaaggtggagacactgtaggatatataatggcagttgaagaat
ID NO: 223)      tgtctagagtttgtggtactacaggagttatattatcagctcatacatctcttggctcatggcctatatatcaat
                 atggtaatgaagaacaaaaacaaaaattcttaagaccactagcaagtggagaaaaattaggagcatttgg
                 tcttactgagcctaatgctggtacagatgcgtctggccaacaaacaactgctgttttagacggggatgaat
                 acatacttaatggctcaaaaatatttataacaaacgcaatagctggtgacatatatgtagtaatggcaatgac
                 tgataaatctaaggggaacaaaggaatatcagcatttatagttgaaaaaggaactcctgggtttagctttgg
                 agttaaagaaaagaaaatgggtataagaggttcagctacgagtgaattaatatttgaggattgcagaatac
                 ctaaagaaaatttacttggaaaagaaggtcaaggatttaagatagcaatgtctactcttgatggtggtagaa
                 ttggtatagctgcacaagctttaggtttagcacaaggtgctcttgatgaaactgttaaatatgtaaaagaaag
                 agtacaatttggtagaccattatcaaaattccaaaatacacaattccaattagctgatatggaagttaaggta
                 caagcggctagacaccttgtatatcaagcagctataaataaagacttaggaaaaccttatggagtagaag
                 cagcaatggcaaaattatttgcagctgaaacagctatggaagttactacaaaagctgtacaacttcatgga
                 ggatatggatacactcgtgactatccagtagaaagaatgatgagagatgctaagataactgaaatatatga
                 aggaactagtgaagttcaaagaatggttatttcaggaaaactattaaaatagtaagaaggagatatacatat
                 ggaggaaggatttatgaatatagtcgtttgtataaaacaagttccagatacaacagaagttaaactagatcc
                 taatacaggtactttaattagagatggagtaccaagtataataaaccctgatgataaagcaggtttagaaga
                 agctataaaattaaaagaagaaatgggtgctcatgtaactgttataacaatgggacctcctcaagcagatat
                 ggctttaaaagaagctttagcaatgggtgcagatagaggtatattattaacagatagagcatttgcgggtg
                 ctgatacttgggcaacttcatcagcattagcaggagcattaaaaaatatagattttgatattataatagctgg
                 aagacaggcgatagatggagatactgcacaagttggacctcaaatagctgaacatttaaatcttccatcaa
                 taacatatgctgaagaaataaaaactgaaggtgaatatgtattagtaaaaagacaatttgaagattgttgcc
                 atgacttaaaagttaaaatgccatgccttataacaactcttaaagatatgaacacaccaagatacatgaaag
                 ttggaagaatatatgatgctttcgaaaatgatgtagtagaaacatggactgtaaaagatatagaagttgacc
                 cttctaatttaggtcttaaaggttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagctggta
                 caatatacaatgaagatgcgaaaacatcagctggaattatcatagataaattaaaagagaagtatatcatat
                 aataagaaggagatatacatatgggtaacgttttagtagtaatagaacaaagagaaaatgtaattcaaact
                 gtttctttagaattactaggaaaggctacagaaatagcaaaagattatgatacaaaagtttctgcattactttt
                 aggtagtaaggtagaaggtttaatagatacattagcacactatggtgcagatgaggtaatagtagtagatg
                 atgaagctttagcagtgtatacaactgaaccatatacaaaagcagcttatgaagcaataaaagcagctgac
                 cctatagttgtattatttggtgcaacttcaataggtagagatttagcgcctagagtttctgctagaatacatac
                 aggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaaattattattaatgacaagacctgcc
                 tttggtggaaatataatggcaacaatagtttgtaaagatttcagacctcaaatgtctacagttagaccaggg
                 gttatgaagaaaaatgaacctgatgaaactaaagaagctgtaattaaccgtttcaaggtagaatttaatgat
                 gctgataaattagttcaagttgtacaagtaataaaagaagctaaaaaacaagttaaaatagaagatgctaa
                 gatattagtttctgctggacgtggaatgggtggaaaagaaaacttagacatactttatgaattagctgaaatt
                 ataggtggagaagtttctggttctcgtgccactatagatgcaggttggttagataaagcaagacaagttggt
                 caaactggtaaaactgtaagaccagacctttatatagcatgtggtatatctggagcaatacaacatatagct
                 ggtatggaagatgctgagtttatagttgctataaataaaaatccagaagctccaatatttaaatatgctgatgt
                 tggtatagttggagatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaaaagaaaaaggt
                 gaagttttagctaactaataagaaggagatatacatatgagagaagtagtaattgccagtgcagctagaac
                 agcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaa
                 gaagctataaaaagagctaacataactccagatatgatagatgaatctcttttagggggagtacttacagc
                 aggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctat
                 gactataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgc
                 tgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggt
                 gcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca
                 catgggtattactgctgaaaacatagcagagcaatggaatataactagagaagaacaagatgaattagct
                 cttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgtt
                 ataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggaga
                 aacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatg
                 atggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaact
                 atagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaa
                 gctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatc
                 tgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctatagga
                 catccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaa
                 aactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaagagatagtaagaagg
                 agatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaagt
                 tgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaa
                 tttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttca
                 actactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatataaaga
                 aagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatcta
                 taacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctttaatccagttcctatg
                 atgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaag
                 agtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgat
                 aaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattag
                 gagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagctataatgaa
                 cgttttatatactgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatca
                 attaggaagaaaaactaagataggattctatgattataataaataataagaaggagatatacatatgagtac
                 aagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatag
                 acctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatg
                 atgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagatattgcataca
                 tgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaa
                 tagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatg
                 tgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatat
                 ggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaag
                 ttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaa
                 gaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata
                 caacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggtctttgtttttcaacta
                 aagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataaga
                 aggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatat
                 cagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaat
                 gccattttagtaggagatatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaatta
                 tgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagc
                 cgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtct
                 tagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactg
                 acgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagca
                 cattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaa
                 agatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggt
                 gggccttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcag
                 gacgagctgatatattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttctcaa
                 aatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaa
                 gaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaaggagatatacat
                 atgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaatagctgtatttgataatga
                 ggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgacca
                 atttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattagatg
                 ctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattga
                 agatttaaaagtgggagttttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggt
                 gaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttc
                 tggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatg
                 ctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttg
                 gagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaa
                 agaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaatt
                 aaaaagaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgctagagaagttgaag
                 aaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaa
                 taggagctagtgctgcagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaa
                 aaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatga
                 aatgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatgataactaa
                 taa
Nucleotide       ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtagaatagcaatgaacgattat
sequences of     ccctatcaagcattctgactgataattgctcacacgaattcattaaagaggagaaaggtaccatgatcgtaa
pLogic046-       aacctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatc
oxyS-butyrate    agattgaatataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttc
construct (SEQ   tggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacggggctgc
ID NO: 224)      gaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaataat
                 ttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcag
                 acgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag
                 cttggccagcccagtacgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaa
                 aaacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaa
                 atgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaagcagc
                 tgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagctaccc
                 aagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctc
                 aacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgc
                 cgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaag
                 gttgtattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgat
                 gaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgttg
                 atggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgatttcttag
                 ctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgtatctgataag
                 aaggagatatacatatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggag
                 gagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaa
                 cataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagc
                 aagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtggtt
                 ctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttggtgga
                 gctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgct
                 tttgttgattcaatgataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaac
                 atagcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctg
                 aaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtga
                 cactgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctg
                 catttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagt
                 aatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctg
                 gtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatg
                 actattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagagactt
                 aaatatagatatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatgctcagg
                 agcaagaatacttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgt
                 ataggcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagc
                 tgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaa
                 agagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagttactaag
                 ggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagattta
                 aaagatatggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagttactag
                 atgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttcta
                 ctaagcgcccagataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttata
                 agtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagt
                 agatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata
                 tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatggg
                 accactagcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttgga
                 gatactaaatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaag
                 ataggattctatgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtttatga
                 gaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaat
                 aaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaata
                 ttgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagc
                 tgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagc
                 tgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgct
                 aaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaaggcttac
                 aagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctg
                 aaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaag
                 ataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgat
                 ataaatactggaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaat
                 gtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgagaa
                 gttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagata
                 aagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatata
                 gaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaag
                 atttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggct
                 tagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaa
                 gtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagct
                 cctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatataagtgaa
                 ccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaa
                 gaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataa
                 tgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggacgagctgatatattatta
                 gccccagatattgaaggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggag
                 ttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactca
                 atagctttaggtgttttaatggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaa
                 atcttaacaataaatcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaa
                 actttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaa
                 gtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggag
                 gacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagtt
                 ttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttc
                 atacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagta
                 gagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaa
                 atatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaa
                 atagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaagaagtggtggactaccagt
                 aggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaa
                 tggcggactagttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatg
                 aaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgcagttc
                 ttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgc
                 agatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggt
                 ggacttagagttttaactggtgaagaagaggctcaagtttatgataactaataa
Nucleotide       ctcgagatgctagcaattgtgagcggataacaattgacattgtgagcggataacaagatactgagcacat
sequences of     cagcaggacgcactgaccttaattaaaagaattcattaaagaggagaaaggtaccatgaatattcgtgatc
pZA22-oxyR       ttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgccacgttagccag
construct (SEQ   ccgacgcttagcgggcaaattcgtaagctggaagatgagctgggcgtgatgttgctggagcggaccag
ID NO: 225)      ccgtaaagtgttgttcacccaggcgggaatgctgctggtggatcaggcgcgtaccgtgctgcgtgaggt
                 gaaagtccttaaagagatggcaagccagcagggcgagacgatgtccggaccgctgcacattggtttgat
                 tcccacagttggaccgtacctgctaccgcatattatccctatgctgcaccagacctttccaaagctggaaat
                 gtatctgcatgaagcacagacccaccagttactggcgcaactggacagcggcaaactcgattgcgtgat
                 cctcgcgctggtgaaagagagcgaagcattcattgaagtgccgttgtttgatgagccaatgttgctggcta
                 tctatgaagatcacccgtgggcgaaccgcgaatgcgtaccgatggccgatctggcaggggaaaaactg
                 ctgatgctggaagatggtcactgtttgcgcgatcaggcaatgggtttctgttttgaagccggggcggatga
                 agatacacacttccgcgcgaccagcctggaaactctgcgcaacatggtggcggcaggtagcgggatca
                 ctttactgccagcgctggctgtgccgccggagcgcaaacgcgatggggttgtttatctgccgtgcattaa
                 gccggaaccacgccgcactattggcctggtttatcgtcctggctcaccgctgcgcagccgctatgagca
                 gctggcagaggccatccgcgcaagaatggatggccatttcgataaagttttaaaacaggcggtttaagga
                 tcccatggtacgcgtgctagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt
                 atctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctaggggatatattcc
                 gcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacgg
                 ggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagcc
                 gtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaac
                 ccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgccttt
                 cggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtag
                 gcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggta
                 actatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatt
                 tagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgct
                 cctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaag
                 gcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatc
                 agataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatat
                 aagttgttactagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatc
                 cagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctctcgaaccccagagtc
                 ccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgatacc
                 gtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacg
                 ctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccatttt
                 ccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgc
                 gccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcga
                 caagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggt
                 agccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaag
                 gtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgac
                 aacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcc
                 tgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacag
                 ccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccac
                 ccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctctt
                 gatcagatcttgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggc
                 ttcccaaccttaccagagggcgccccagctggcaattccgacgtctaagaaaccattattatcatgacatt
                 aacctataaaaataggcgtatcacgaggccctttcgtcttcac
Nucleotide       gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatca
sequences of     attcaaggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatg
pLogic031-tet-   gcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac
butyrate         ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaa
construct (SEQ   aggctaattgattttcgagagtttcatactgtttttctgtaggccgt gtacctaaatgtacttttgctccatcgcg
ID NO: 226) The  atgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaag
sequence         ggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattatta
encoding TetR is catgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattcc
underlined, and  gacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt
the overlapping
tetR/tetA        attttgtttaactttaagaaggagatatacatatggatttaaattctaaaaaatatcagatgcttaaagagctat
promoters are    atgtaagcttcgctgaaaatgaagttaaacctttagcaacagaacttgatgaagaagaaagatttccttatg
                 aaacagtggaaaaaatggcaaaagcaggaatgatgggtataccatatccaaaagaatatggtggagaa
                 ggtggagacactgtaggatatataatggcagttgaagaattgtctagagtttgtggtactacaggagttata
                 ttatcagctcatacatctcttggctcatggcctatatatcaatatggtaatgaagaacaaaaacaaaaattctt
                 aagaccactagcaagtggagaaaaattaggagcatttggtcttactgagcctaatgctggtacagatgcgt
                 ctggccaacaaacaactgctgttttagacggggatgaatacatacttaatggctcaaaaatatttataacaa
                 acgcaatagctggtgacatatatgtagtaatggcaatgactgataaatctaaggggaacaaaggaatatc
                 agcatttatagttgaaaaaggaactcctgggtttagctttggagttaaagaaaagaaaatgggtataagag
                 gttcagctacgagtgaattaatatttgaggattgcagaatacctaaagaaaatttacttggaaaagaaggtc
                 aaggatttaagatagcaatgtctactcttgatggtggtagaattggtatagctgcacaagctttaggtttagc
                 acaaggtgctcttgatgaaactgttaaatatgtaaaagaaagagtacaatttggtagaccattatcaaaattc
                 caaaatacacaattccaattagctgatatggaagttaaggtacaagcggctagacaccttgtatatcaagc
                 agctataaataaagacttaggaaaaccttatggagtagaagcagcaatggcaaaattatttgcagctgaaa
                 cagctatggaagttactacaaaagctgtacaacttcatggaggatatggatacactcgtgactatccagta
                 gaaagaatgatgagagatgctaagataactgaaatatatgaaggaactagtgaagttcaaagaatggttat
                 ttcaggaaaactattaaaatagtaagaaggagatatacatatggaggaaggatttatgaatatagtcgtttgt
                 ataaaacaagttccagatacaacagaagttaaactagatcctaatacaggtactttaattagagatggagta
                 ccaagtataataaaccctgatgataaagcaggtttagaagaagctataaaattaaaagaagaaatgggtg
                 ctcatgtaactgttataacaatgggacctcctcaagcagatatggctttaaaagaagctttagcaatgggtg
                 cagatagaggtatattattaacagatagagcatttgcgggtgctgatacttgggcaacttcatcagcattag
                 caggagcattaaaaaatatagattttgatattataatagctggaagacaggcgatagatggagatactgca
                 caagttggacctcaaatagctgaacatttaaatcttccatcaataacatatgctgaagaaataaaaactgaa
                 ggtgaatatgtattagtaaaaagacaatttgaagattgttgccatgacttaaaagttaaaatgccatgccttat
                 aacaactcttaaagatatgaacacaccaagatacatgaaagttggaagaatatatgatgctttcgaaaatg
                 atgtagtagaaacatggactgtaaaagatatagaagttgacccttctaatttaggtcttaaaggttctccaac
                 tagtgtatttaaatcatttacaaaatcagttaaaccagctggtacaatatacaatgaagatgcgaaaacatca
                 gctggaattatcatagataaattaaaagagaagtatatcatataataagaaggagatatacatatgggtaac
                 gttttagtagtaatagaacaaagagaaaatgtaattcaaactgtttctttagaattactaggaaaggctacag
                 aaatagcaaaagattatgatacaaaagtttctgcattacttttaggtagtaaggtagaaggtttaatagatac
                 attagcacactatggtgcagatgaggtaatagtagtagatgatgaagctttagcagtgtatacaactgaac
                 catatacaaaagcagcttatgaagcaataaaagcagctgaccctatagttgtattatttggtgcaacttcaat
                 aggtagagatttagcgcctagagtttctgctagaatacatacaggtcttactgctgactgtacaggtcttgca
                 gtagctgaagatacaaaattattattaatgacaagacctgcctttggtggaaatataatggcaacaatagttt
                 gtaaagatttcagacctcaaatgtctacagttagaccaggggttatgaagaaaaatgaacctgatgaaact
                 aaagaagctgtaattaaccgtttcaaggtagaatttaatgatgctgataaattagttcaagttgtacaagtaat
                 aaaagaagctaaaaaacaagttaaaatagaagatgctaagatattagtttctgctggacgtggaatgggtg
                 gaaaagaaaacttagacatactttatgaattagctgaaattataggtggagaagtttctggttctcgtgccac
                 tatagatgcaggttggttagataaagcaagacaagttggtcaaactggtaaaactgtaagaccagaccttt
                 atatagcatgtggtatatctggagcaatacaacatatagctggtatggaagatgctgagtttatagttgctat
                 aaataaaaatccagaagctccaatatttaaatatgctgatgttggtatagttggagatgttcataaagtgcttc
                 cagaacttatcagtcagttaagtgttgcaaaagaaaaaggtgaagttttagctaactaataagaaggagat
                 atacatatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagcattta
                 aatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcc
                 agatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaagacaaat
                 agcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtggttctggattaag
                 atctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca
                 tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattca
                 atgataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagag
                 caatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctc
                 aagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagta
                 gataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaa
                 gatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaa
                 gaaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccct
                 aaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattgaagat
                 atagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatatagatat
                 gaataaagttaatgttaatggtggagcaatagctataggacatccaataggatgctcaggagcaagaata
                 cttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtg
                 gaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagctgtaataggta
                 gtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaac
                 tcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagttactaagggaaaaatgg
                 atgaagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaaagatatgga
                 tttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgta
                 aagaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcgccc
                 agataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcagtt
                 aacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga
                 atctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatatatgcagatggtgt
                 tgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccactagcatta
                 ggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttggagatactaaatata
                 gacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattctatg
                 attataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtttatgagaatgtagctgtt
                 gaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagac
                 tttagaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacagggga
                 aggaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattt
                 tagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacg
                 gatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgctaaagctaaattt
                 ggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaaggcttacaagattggttg
                 gaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagg
                 gctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaagataatagctaa
                 aaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactgg
                 aatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgtt
                 gaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgagaagttttgaagaag
                 taattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagataaagaagttttaa
                 tggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatatagaaaagactaa
                 agaaattgcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaag
                 catctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggcttagtagacacat
                 caataatactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcag
                 tatttgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagctcctgatacaaat
                 actaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatataagtgaaccaaaagttgct
                 gcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaa
                 atgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataatgcagtatctttag
                 aagcagctaaacataaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagatattg
                 aaggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggct
                 aaagcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactcaatagctttaggtgtt
                 ttaatggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaat
                 cctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaactttaagacattct
                 tcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagc
                 tctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggaggacttcttaaaccta
                 taaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggagaacacg
                 cttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttcatacatagtagac
                 cctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtag
                 tacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatata
                 aatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaaatagtagatgttg
                 caaacgcattagatggagaaggacctttctctccagaaagaagtggtggactaccagtaggtgcattagt
                 aaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactag
                 ttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaa
                 ttagtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgcagttcttaagggagat
                 gtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagtta
                 aatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggacttagagt
                 tttaactggtgaagaagaggctcaagtttatgataactaataa
Nucleotide       gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatca
sequences of     attcaaggccgaataagaaggctggctctgcaccaggtgatcaaataattcgatagatgtcgtaataatg
pLogic046-tet-   gcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac
butyrate         ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaa
construct (SEQ   aggctaattgattacgagagatcatactgatactgtaggccgtgtacctaaatgtacattgctccatcgce
ID NO: 227) The  atgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaag
sequence         ggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattatta
encoding TetR is catgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattcc
underlined, and  gacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt
the overlapping
tetR/tetA        attttgtttaactttaagaaggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaac
promoters are    gcccatcctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgc
                 agaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggttacggcctg
                 gcgagccgcattactgctgcgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggtt
                 cagaaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggcaaaacgcgagg
                 gtctttatagcgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaagc
                 caaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgatac
                 aggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatccgttta
                 ctggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagccgccactgttaaagtt
                 atggggggtgaagattgggaacgttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgt
                 attaccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaag
                 gccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgt
                 gagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag
                 cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccg
                 agcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgattgggag
                 ttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatc
                 tctcactgacttagcggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatg
                 aagcggaagttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc
                 cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagttaggg
                 gtaacagcagctaaagaagctataaaaagagctaacataactccagatatgatagatgaatctcttttagg
                 gggagtacttacagcaggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagt
                 agaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttat
                 agcattaggtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaa
                 gtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatcagacat
                 atttaataactatcacatgggtattactgctgaaaacatagcagagcaatggaatataactagagaagaac
                 aagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaaga
                 aatagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctgg
                 cactacaatggagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatg
                 catcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaata
                 gagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttc
                 cagcaactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgagg
                 catttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtgga
                 gcaatagctataggacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaag
                 agaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaag
                 agatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgt
                 acaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgttta
                 gctttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaata
                 ttaagtcatgttagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaa
                 gacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaa
                 atacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttct
                 ttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacag
                 tatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaa
                 tacttatacctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatga
                 agctatgaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgt
                 tttagctataatgaacgttttatatactgaatttggagatactaaatatagacctcatccacttttagctaaaatg
                 gttagagctaatcaattaggaagaaaaactaagataggattctatgattataataaataataagaaggagat
                 atacatatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacag
                 tgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgt
                 agatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagc
                 agatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttg
                 gagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaa
                 cttgcaatggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttgga
                 ataactccaggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaa
                 tctttacaggtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagcca
                 gacattttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagatact
                 ctaaagaagcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggt
                 ctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataa
                 aagggtaataagaaggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagagg
                 acctaaaactatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaag
                 aaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgcaaaaagcatagacat
                 ggatatcgaaaattatgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagttt
                 cacaaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaat
                 aaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgataga
                 ttatttttcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatg
                 cttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagta
                 aatccaaaaatgaaagatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaa
                 ggttgtatggttggtgggccttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataa
                 atcatcctgtagcaggacgagctgatatattattagccccagatattgaaggtggtaacatattatataaagc
                 tttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttcta
                 gagcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataa
                 gaaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaata
                 gctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaatatga
                 gaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaa
                 acatctgaattagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaa
                 gtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgcttcaaacctaggtggaataatag
                 caaaacaaataggtgaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaag
                 atgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaat
                 agcaagaagatatgctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgggtg
                 gaggagtttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaagga
                 cctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatat
                 actcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgc
                 tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatc
                 aaatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatattattaactggtg
                 gaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttat
                 ccaggtgaagatgaaatgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctca
                 agtttatgataactaataa

In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.

In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB. TesB is a thioesterase found in E. Coli that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk.

In one embodiment, the tesB cassette is placed under the control of a FNR regulatory region selected from any of the sequences in Table 6. In an alternate embodiment, the tesB cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR. In yet another embodiment, the tesB cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR. In certain constructs, the different described butyrate gene cassettes are each placed under the control of a tetracycline-inducible or constitutive promoter. For example, genetically engineered Nissle are generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB expressed under the control of a nitric oxide-responsive regulatory element. SEQ ID NO: 228 comprises a reverse complement of the nsrR repressor gene from Neisseria gonorrhoeae (underlined), intergenic region containing divergent promoters controlling nsrR and the butyrogenic gene cassette and their respective RBS (bold), and the butyrate genes (ter-thiA-hbd-crt-tesB) separated by RBS.

TABLE 37
SEQ ID NO: 228
ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggctt
gttgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgc
accgccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgg
gcccatacactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatatt
gatgcgttcgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtg
caagaacccgcctttgaccagcgcggtaaccactttcatcaaatggcttttggaaat
gccgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggt
gtagatgaggacgcgcagcccgtagtcggtatgttgggtcagatacat              acaacctcc
ttagtacatgcaaaattatttctagagcaacatacgagccggaagcataaagtgtaa
agcctggggtgcctaatgagttgagttgaggaattataacaggaagaaatattcctc
atacgcttgtaattcctctatggttgttgacaattaatcatcggctcgtataatgta
taacattcatattttgtgaattttaaactctagaaataattttgtttaactttaaga
aggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgc
ccatcctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacg
cattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg
ctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacggggc
tgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacacc
gggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatag
cgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgagga
agccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagt
acgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaa
aacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgc
ggaaccagcaaatgacgaggaagcagccgccactgttaaagttatggggggtgaaga
ttgggaacgttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgtat
taccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaagg
cacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctcaacaaaga
gaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaag
cgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaaga
gaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcct
gtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatga
ttgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcac
gggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgatttcttagc
tagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcga
ccgtatctgataagaaggagatatacatatgagagaagtagtaattgccagtgcagc
tagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagtt
aggggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgat
agatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaag
acaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatat
agtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattagg
tgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt
agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaat
gataaaagatggattatcagacatatttaataactatcacatgggtattactgctga
aaacatagcagagcaatggaatataactagagaagaacaagatgaattagctcttgc
aagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagt
tcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatat
taagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaaga
tggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagt
agtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagt
ttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagc
aactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttga
agctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatataga
tatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatg
ctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaa
aactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaa
gagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactat
gggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagag
tagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaa
gttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgt
tagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatc
tgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaa
agaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttc
ttctactaagcgcccagataaagttataggaatgcatttctttaatccagttcctat
gatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatac
agtatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcc
tggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata
tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaa
ccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagc
tataatgaacgttttatatactgaatttggagatactaaatatagacctcatccact
tttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattcta
tgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagt
ttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatag
acctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatt
tgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaa
ggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgc
taaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa
agtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaat
ggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaac
tcttggaataactccaggatatggaggaactcaaaggcttacaagattggttggaat
ggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctga
aaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttga
gaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaaga
agcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaa
tttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgt
tgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgAG
TCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAATTGAGGAAGGACT
CTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTGGCGGCCAGGTCGT
GGGTCAGGCCTTGTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC
GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGT
CGAAACGCTGCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAA
CGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGA
ACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAACGCA
AATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCTG
CGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGT
CGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCT
GCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTGCCGGTAGC
TCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGA
CCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGT
GGAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACCCA
AGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATCACAATta
a

Example 11

Construction of Vectors for Overproducing Butyrate Using a tet-Inducible Promoter

To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile (bcd, etfB, etfA, thiA, hbd, crt, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen, Cambridge, Mass.) and cloned into vector pBR to create pLogic. As synthesized, the genes were placed under control of a tetracycline-inducible promoter, with the tet repressor (tetR) expressed constitutively, divergent from the tet-inducible synthetic butyrate operon. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a base pair ribosome binding site derived from the T promoter.

The gene products of bcd-etfA-etfB form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. Because an effective probiotic should be able to function in an oxygen-limited environment (e.g. the mammalian gut), and because it has been shown that a single gene from Treponema denticola can functionally replace this three gene complex in an oxygen-independent manner (trans--enoynl-CoA reductase; ter), we created a second plasmid capable of butyrate production in E. coli . Inverse PCR was used to amplify the entire sequence of pLogic outside of the bcd-etfA-etfB region. The ter gene was codon optimized for E. coli codon usage using Integrated DNA technologies online codon optimization tool (https://www.idtdna.com/CodonOpt), synthesized (Genewiz, Cambridge, Mass.), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic.

Example 12

Transforming E. coli

Each plasmid is transformed into E. coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli Nissle or E. coli DH5a is diluted 1:100 in 5 mL of lysogeny broth (LB) and grown until it reached an OD₆₀₀ of 0.4-0.6. The cell culture medium contains a selection marker, e.g., ampicillin, that is suitable for the plasmid. The E. coli cells are then centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are finally resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of one of the above plasmids is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. One mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing ampicillin and incubated overnight.

In alternate embodiments, the butyrate cassette can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are provided herein. To create a vector capable of integrating the synthesized butyrate cassette construct into the chromosome, Gibson assembly was first used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the butyrate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20 ug/mL at 37 degrees C. Growth at 37 degrees C. also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac−).

Example 13

Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 14

Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 15

Production of Butyrate in Recombinant E. coli Using Tet-Inducible Promoter

FIG. 2 shows butyrate cassettes described above under the control of a tet-inducible promoter. Production of butyrate is assessed using the methods described below in Example 21. The tet-inducible cassettes tested include (1) tet-butyrate cassette comprising all eight genes (pLOGIC031); (2) tet-butyrate cassette in which the ter is substituted (pLOGIC046) and (3) tet-butyrate cassette in which tesB is substituted in place of pbt and buk genes.

FIG. 6A shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl CoA).

Overnight cultures of cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of 100 ng/m1 to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at OD600=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS. FIG. 6B shows butyrate production in strains comprising a tet-butyrate cassette having ter substitution (pLOGIC046) or the tesB substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production.

FIG. 7 shows the BW25113 strain of E. Coli, which is a common cloning strain and the background of the KEIO collection of E. Coli mutants. NuoB mutants having NuoB deletion were obtained. NuoB is a protein complex involved in the oxidation of NADH during respiratory growth (form of growth requiring electron transport). Preventing the coupling of NADH oxidation to electron transport allows an increase in the amount of NADH being used to support butyrate production. FIG. 7 shows that compared with wild-type Nissle, deletion of NuoB results in grater production of butyrate.

TABLE 38
pLOGIC046-tesB-butyrate (SEQ ID NO: 229)
pLOGIC046-tesB-butyrate: SEQ ID NO: 229
gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgttt
ttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctgcaccttggt
gatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtt
tccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaa
atgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata
aaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaa
tgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttatt
acgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctat
ctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac
aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgat
tccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctag
acatcattaattcctaatttttgttgacactctatcattgatagagttattttaccact
ccctatcagtgatagagaaaagtgaactctagaaataattttgtttaactttaagaagg
agatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatc
ctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattacc
gcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaa
tggttacggcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcg
gcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaat
aatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacgg
cgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggta
tcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgataca
ggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagt
agatccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaag
cagccgccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctg
tcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccc
tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctgg
aggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtg
aataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgc
cagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatca
cgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaa
aatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgc
gttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtacc
gccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaa
gttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaat
tgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcag
cggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcca
gatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatat
agcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataa
atatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcatta
ggtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt
agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatga
taaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaac
atagcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtca
aaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttg
ttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggc
actacaatggagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttac
tgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaag
aaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagct
ggtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaagctttaga
agctgctaatatgactattgaagatatagatttagttgaagctaatgaggcatttgctg
cccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaat
ggtggagcaatagctataggacatccaataggatgctcaggagcaagaatacttactac
acttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtatag
gcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatacatatg
aaattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaag
ttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgtttag
ctttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctaca
aaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaaagatat
ggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagt
tactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatct
ataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctt
taatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaag
ttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgta
tctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgt
tggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattag
gagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgtt
ttagctataatgaacgttttatatactgaatttggagatactaaatatagacctcatcc
acttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattct
atgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtt
tatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc
taaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtag
atattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcattt
gtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattt
tagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatag
ctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatata
agaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactcc
aggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaat
taatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaat
agagtcgttgagccagacattttaatagaagaagttgagaaattagctaagataatagc
taaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaa
ctgatataaatactggaatagatatagaatctaatttatttggtctttgtttttcaact
aaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataaa
agggtaataagaaggagatatacatatgAGTCAGGCGCTAAAAAATTTACTGACATTGT
TAAATCTGGAAAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTTA
CGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACCGT
CCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTA
AGAAGCCGATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGCGCCCGC
CGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGC
ACCAGAAGCGGGTTTCGAACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCC
TCCCTTCGGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAA
GATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACT
GAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGC
CGGATGACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTG
CCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCAC
CATTGACCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATA
GCGTGGAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACC
CAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATCACAATta
a

Example 16

Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N₂, 5% CO₂, 5%H₂). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

In an alternate embodiment, overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Genetically engineered Nissle comprising pLogic031-nsrR-norB-butyrate operon construct; SYN507) or (pLogic046-nsrR-norB-butyrate operon construct; SYN--508) produce significantly more butyrate as compared to wild-type Nissle.

Genetically engineered Nissle were generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB (SEQ ID NO: 15) expressed under the control of a tetracycline promoter (pLOGIC046-tesB-butyrate; SEQ ID NO: 208). SEQ ID NO: 208 comprises a reverse complement of the tetR repressor (underlined), an intergenic region containing divergent promoters controlling tetR and the butyrate operon and their respective RBS (bold), and the butyrate genes (ter-thiAl-hbd-crt2-tesB) separated by RBS.

Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, anhydrous tetracycline (ATC) was added to cultures at a final concentration of 100 ng/mL to induce expression of butyrate genes from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Replacement of pbt and buk with tesB leads to greater levels of butyrate production.

FIG. 8C shows butyrate production in strains comprising an FNR-butyrate cassette SYN501 (having the ter substitution) in the presence/absence of glucose and oxygen. FIG. 8C shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containing no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicated time pints and supernatant fractions were assessed for butyrate concentration using LC-MS. These data show that SYN501 requires glucose for butyrate production and that in the presence of glucose butyrate production can be enhanced under anaerobic conditions when under the control of the anaerobic FNR-regulated ydfZ promoter.

TABLE 39
Butyrate cassette sequences
		SEQ ID
Description	Sequence	NO
ydfZ + RBS	CATTTCCTCTCATCCCATCCGGGGTGAGAGT	SEQ ID
(RBS is bolded)	CTTTTCCCCCGACTTATGGCTCATGCATGCAT	NO: 230
	CAAAAAAGATGTGAGCTTGATCAAAAACAA
	AAAATATTTCACTCGACAGGAGTATTTATAT
	TGCGCCCGGATCCCTCTAGAAATAATTTTGT
	TTAACTTTAAGAAGGAGATATACAT
First RBS (in	TTTGTTTAACTTTAAGAAGGAGA	SEQ ID
ydfZ = RBS)		NO: 231
Internal RBS	taagaaggagatatacat	SEQ ID
between genes		NO: 211
Butyrate cassette	CATTTCCTCTCATCCCATCCGGGGTGAGAGT	SEQ ID
under the control	CTTTTCCCCCGACTTATGGCTCATGCATGCAT	NO: 232
of the ydfZ	CAAAAAAGATGTGAGCTTGATCAAAAACAA
promoter	AAAATATTTCACTCGACAGGAGTATTTATAT
(uppercase: ydfZ	TGCGCCCGGATCCCTCTAGAAATAATTTTGT
promoter, with	TTAACTTTAAGAAGGAGATATACATatgatcgt
RBS in bold;	aaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggct
lower case:	gcaagaagggagtggaagatcagattgaatataccaagaaacgcattac
coding regions	cgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgctt
in the following	ggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcgg
order: ter, thiA,	atacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaa
hbd, crt2, pbt,	accaaatatggtacaccgggatggtacaataatttggcatttgatgaagcg
buk, separated	gcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttc
by internal RBS	agacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtat
(uppercase and	caaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcct
underlined)	gatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttc
	acaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccg
	cggaaccagcaaatgacgaggaagcagccgccactgttaaagttatgg
	ggggtgaagattgggaacgttggattaagcagctgtcgaaggaaggcct
	cttagaagaaggctgtattaccttggcctatagttatattggccctgaagct
	acccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacc
	tggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtg
	ccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaat
	cccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaa
	gggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagc
	gcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattc
	gcattgatgattgggagttagaagaagacgtccagaaagcggtatccgc
	gttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttag
	cggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtat
	taattatgaagcggaagttgaacgcttcgaccgtatctgaTAAGAA
	GGAGATATACATatgagagaagtagtaattgccagtgcagct
	agaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggta
	gagttaggggtaacagcagctaaagaagctataaaaagagctaacataa
	ctccagatatgatagatgaatctcttttagggggagtacttacagcaggtct
	tggacaaaatatagcaagacaaatagcattaggagcaggaataccagta
	gaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgt
	ttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttg
	gtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagata
	tggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatgga
	ttatcagacatatttaataactatcacatgggtattactgctgaaaacatagc
	agagcaatggaatataactagagaagaacaagatgaattagctcttgcaa
	gtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaa
	atagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaa
	gatgaatatattaagcctggcactacaatggagaaacttgctaagttaaga
	cctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaata
	aatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaa
	ctaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttg
	accctaaaataatgggatatggaccagttccagcaactaaaaaagctttag
	aagctgctaatatgactattgaagatatagatttagttgaagctaatgaggc
	atttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaata
	aagttaatgttaatggtggagcaatagctataggacatccaataggatgct
	caggagcaagaatacttactacacttttatatgaaatgaagagaagagatg
	ctaaaactggtcttgctacactttgtataggcggtggaatgggaactacttt
	aatagttaagagatagTAAGAAGGAGATATACATatgaa
	attagctgtaataggtagtggaactatgggaagtggtattgtacaaactttt
	gcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctata
	gataaatgtttagctttattagataaaaatttaactaagttagttactaaggga
	aaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaact
	actaattatgaagatttaaaagatatggatttaataatagaagcatctgtaga
	agacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaa
	gaagatactatcttggcaacaaatacttcatcattatctataacagaaatag
	cttcttctactaagcgcccagataaagttataggaatgcatttctttaatcca
	gttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagt
	tacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtaga
	tgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaa
	tgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaataga
	tgaagctatgaaattaggagcaaaccatccaatgggaccactagcattag
	gtgatttaatcggattagatgttgttttagctataatgaacgttttatatactga
	atttggagatactaaatatagacctcatccacttttagctaaaatggttagag
	ctaatcaattaggaagaaaaactaagataggattctatgattataataaata
	aTAAGAAGGAGATATACATatgagtacaagtgatgttaa
	agtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaa
	atgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaa
	ctttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatatt
	gacaggggaaggaaaggcatttgtagctggagcagatattgcatacatg
	aaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaag
	cttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacg
	gatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaat
	tgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataact
	ccaggatatggaggaactcaaaggcttacaagattggttggaatggcaa
	aagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctg
	aaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaag
	aagttgagaaattagctaagataatagctaaaaatgctcagcttgcagtta
	gatactctaaagaagcaatacaacttggtgctcaaactgatataaatactg
	gaatagatatagaatctaatttatttggtctttgatttcaactaaagaccaaaa
	agaaggaatgtcagctttcgttgaaaagagagaagctaactttataaaag
	ggtaaTAAGAAGGAGATATACATatgagaagttttgaag
	aagtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcat
	gttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaa
	aaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaatt
	gcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaa
	gatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaag
	ccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagca
	gttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtag
	cagtatttgatgtagagggatatgatagattatttttcgtaactgacgcagct
	atgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgctt
	gcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatat
	gcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaa
	agaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttg
	gtgggccttttgcaattgataatgcagtatctttagaagcagctaaacataa
	aggtataaatcatcctgtagcaggacgagctgatatattattagccccaga
	tattgaaggtggtaacatattatataaagctttggtattcttctcaaaatcaaa
	aaatgcaggagttatagttggggctaaagcaccaataatattaacttctag
	agcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatg
	gcagcaaaggcataaTAAGAAGGAGATATACATatga
	gcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaata
	gctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttca
	gaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaa
	caagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaatt
	agatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtac
	ttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggag
	aacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaaga
	agtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaag
	atgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtaca
	tgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaaca
	agaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttc
	tgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagat
	ggagaaggacctttctctccagaaagaagtggtggactaccagtaggtg
	cattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaa
	gaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgc
	tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattag
	tatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctg
	cagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcata
	ttcaaaaatgtttacagaaatgattgcagatagagttaaatttatagcagatg
	taaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggact
	tagagttttaactggtgaagaagaggctcaagtttatgataactaataa

In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 232 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 232 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 232 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 232 or a functional fragment thereof.

In alternate embodiments, pbt and buk are replaced with TesB (SEQ ID NO: 15)

In some embodiments, the butyrate cassette is driven by an inducible promoter. For example, other FNR promoters can be used in lieu of ydfZ, e.g., in SEQ ID NO: 180-196.

Non-limiting FNR promoter sequences are provided herein. In some embodiments, the genetically engineered bacteria of the invention comprise a butyrate cassette under the control of one or more of promoter sequences found in Table 6, e.g., nirB promoter, ydfZ promoter, nirB promoter fused to a strong ribosome binding site, ydfZ promoter fused to a strong ribosome binding site, fnrS, an anaerobically induced small RNA gene (fnrS promoter), nirB promoter fused to a crp binding site, and fnrS fused to a crp binding site.

In some embodiments, the butyrate cassette is under the control of a promoter which is inducible by metabolites present in the gut. In some embodiments, the butyrate cassette is induced by HE-specific molecules or metabolites indicative of liver damage, e.g., bilirubin. In some embodiments, the butyrate cassette is placed under the control of promoter, which is inducible by inflammation or an inflammatory response (e.g., RNS or ROS promoter).

In some embodiments, the genetically engineered bacteria comprise a butyrate cassette driven by a promoter induced by a molecule or metabolite. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein.

In some embodiments, the butyrate cassette is inducible by arabinose and is driven by the AraBAD promoter.

Example 17

Comparison of In Vitro Butyrate Production Efficacy of Chromosomal Insertion and Plasmid-Bearing Engineered Bacterial Strains

The in vitro butyrate production efficacy of engineered bacterial strains harboring a chromosomal insertion of a butyrate cassette was compared to a strain bearing a butyrate cassette on a plasmid. SYN1001 and SYN1002 harbor a chromosomal insertion between the agal/rsml locus of a butyrate cassette (either ter→tesB or ter→pbt-buk, respectively) driven by an fnr inducible promoter. These strains were compared side by side with the low copy plasmid strain SYN501 (Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid) also driven by an fnr inducible promoter. Butyrate levels in the media were measured at 4 and 24 hours post anaerobic induction.

Briefly, 3 ml LB was inoculated with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 dilution into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (4 and 24 h), 120 ul cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 21). Results are depicted in FIG. 11, and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501.

TABLE 40
FRNRs Butyrate Cassette Sequences
Description               Sequence
Pfnrs-ter-thiA1-hbd-ctr2- GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT
tesB                      GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC
SEQ ID NO: 233, e.g.      AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC
integrated into the       AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC
chromosome in             ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA
SYN1001 Pfnrs:            ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG
uppercase; butyrate       AGATATACATatgatcgtaaaacctatggtacgcaacaatatctgcctg
cassette: lower case      aacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatatac
                          caagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaac
                          gttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctg
                          cgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcag
                          aaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggc
                          aaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcagacga
                          gatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatct
                          gatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcatg
                          cacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatc
                          cgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaa
                          gcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaag
                          cagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagtt
                          atattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaagg
                          ccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtc
                          aatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccg
                          taatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaag
                          ggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgt
                          accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgat
                          tgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagt
                          cacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgattt
                          cttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaac
                          gcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc
                          cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttca
                          gcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacat
                          aactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttg
                          gacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaa
                          ccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcat
                          ctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca
                          tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgat
                          gctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca
                          catgggtattactgctgaaaacatagcagagcaatggaatataactagagaaga
                          acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaa
                          ggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgaca
                          ctgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgct
                          aagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcag
                          gaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaaga
                          actaggaatagagcctcagcaactatagtacttatggaacagctggtgagacc
                          ctaaaataatgggatatggaccagaccagcaactaaaaaagcatagaagctgc
                          taatatgactattgaagatatagatttagagaagctaatgaggcatagctgccca
                          atctgtagctgtaataagagacttaaatatagatatgaataaagttaatgaaatggt
                          ggagcaatagctataggacatccaataggatgctcaggagcaagaatacttact
                          acacattatatgaaatgaagagaagagatgctaaaactggtcagctacactagt
                          ataggcggtggaatgggaactacataatagttaagagatagtaagaaggagat
                          atacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca
                          aacattgcaagagtggacatgatgtatgataaagagtagaactcaaggtgctat
                          agataaatgatagcatattagataaaaatttaactaagttagttactaagggaaaa
                          atggatgaagctacaaaagcagaaatattaagtcatgaagacaactactaattat
                          gaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatat
                          aaagaaagatgattcaagttactagatgaattatgtaaagaagatactatcaggc
                          aacaaatacttcatcattatctataacagaaatagatcactactaagcgcccaga
                          taaagttataggaatgcatttattaatccagacctatgatgaaattagagaagtta
                          taagtggtcagttaacatcaaaagttacattgatacagtatttgaattatctaagagt
                          atcaataaagtaccagtagatgtatctgaatctcctggatagtagtaaatagaata
                          cttatacctatgataaatgaagctgaggtatatatgcagatggtgagcaagtaaa
                          gaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccacta
                          gcattaggtgatttaatcggattagatgagattagctataatgaacgattatatact
                          gaataggagatactaaatatagacctcatccactatagctaaaatggttagagct
                          aatcaattaggaagaaaaactaagataggattctatgattataataaataataaga
                          aggagatatacatatgagtacaagtgatgaaaagatatgagaatgtagctgaga
                          agtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgca
                          ataaattcaaagacatagaagaacatatgaagtatagtagatattaataatgatg
                          aaactattgatgagtaatattgacaggggaaggaaaggcatagtagctggagc
                          agatattgcatacatgaaagatttagatgctgtagctgctaaagatatagtatctta
                          ggagcaaaagataggagaaatagaaaatagtaaaaaagtagtgatagctgct
                          gtaaacggatttgattaggtggaggatgtgaacttgcaatggcatgtgatataag
                          aattgcatctgctaaagctaaataggtcagccagaagtaactcaggaataactc
                          caggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagca
                          aaagaattaatattacaggtcaagttataaaagctgatgaagctgaaaaaatagg
                          gctagtaaatagagtcgagagccagacatataatagaagaagagagaaattag
                          ctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata
                          caacaggtgctcaaactgatataaatactggaatagatatagaatctaatttatag
                          gtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaaga
                          gagaagctaacatataaaagggtaataagaaggagatatacatatgagtcagg
                          cgctaaaaaatttactgacattgaaaatctggaaaaaattgaggaaggactattc
                          gcggccagagtgaagatttaggtttacgccaggtgtttggcggccaggtcgtgg
                          gtcaggccagtatgctgcaaaagagaccgtccctgaagagcggctggtacatt
                          cgatcacagctactacttcgccctggcgatagtaagaagccgattatttatgatgt
                          cgaaacgctgcgtgacggtaacagcttcagcgcccgccgggagctgctattca
                          aaacggcaaaccgattattatatgactgcctctaccaggcaccagaagcggga
                          tcgaacatcaaaaaacaatgccgtccgcgccagcgcctgatggcctccatcgg
                          aaacgcaaatcgcccaatcgctggcgcacctgctgccgccagtgctgaaagat
                          aaattcatctgcgatcgtccgctggaagtccgtccggtggagatcataacccact
                          gaaaggtcacgtcgcagaaccacatcgtcaggtgtggatccgcgcaaatggta
                          gcgtgccggatgacctgcgcgttcatcagtatctgctcggttacgcttctgatctta
                          acttcctgccggtagctctacagccgcacggcatcggttttctcgaaccggggat
                          tcagattgccaccattgaccattccatgtggttccatcgcccgtttaatttgaatgaa
                          tggctgctgtatagcgtggagagcacctcggcgtccagcgcacgtggctttgtg
                          cgcggtgagttttatacccaagacggcgtactggttgcctcgaccgttcaggaa
                          ggggtgatgcgtaatcacaattaa
Pfnrs-ter-thiA1-hbd-crt2- GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT
pbt-buk                   GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC
(SEQ ID NO: 234), e.g.    AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC
integrated into the       AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC
chromosome in             ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA
SYN1002 Pfnrs:            ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG
uppercase; butyrate       AGATATACATatgatcgtaaaacctatggtacgcaacaatatctgcctg
cassette: lower case      aacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatatac
                          caagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaac
                          gttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctg
                          cgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcag
                          aaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggc
                          aaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcagacga
                          gatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatct
                          gatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcatg
                          cacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatc
                          cgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaa
                          gcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaag
                          cagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagtt
                          atattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaagg
                          ccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtc
                          aatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccg
                          taatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaag
                          ggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgt
                          accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgat
                          tgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagt
                          cacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgattt
                          cttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaac
                          gcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc
                          cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttca
                          gcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacat
                          aactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttg
                          gacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaa
                          ccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcat
                          ctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca
                          tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgat
                          gctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca
                          catgggtattactgctgaaaacatagcagagcaatggaatataactagagaaga
                          acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaa
                          ggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgaca
                          ctgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgct
                          aagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcag
                          gaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaaga
                          actaggaatagagcctcagcaactatagtacttatggaacagctggtgagacc
                          ctaaaataatgggatatggaccagaccagcaactaaaaaagcatagaagctgc
                          taatatgactattgaagatatagatttagagaagctaatgaggcatagctgccca
                          atctgtagctgtaataagagacttaaatatagatatgaataaagttaatgaaatggt
                          ggagcaatagctataggacatccaataggatgctcaggagcaagaatacttact
                          acacattatatgaaatgaagagaagagatgctaaaactggtcagctacactagt
                          ataggcggtggaatgggaactacataatagttaagagatagtaagaaggagat
                          atacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca
                          aacattgcaagagtggacatgatgtatgataaagagtagaactcaaggtgctat
                          agataaatgatagcatattagataaaaatttaactaagttagttactaagggaaaa
                          atggatgaagctacaaaagcagaaatattaagtcatgaagacaactactaattat
                          gaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatat
                          aaagaaagatgattcaagttactagatgaattatgtaaagaagatactatcaggc
                          aacaaatacttcatcattatctataacagaaatagatcactactaagcgcccaga
                          taaagttataggaatgcatttattaatccagacctatgatgaaattagagaagtta
                          taagtggtcagttaacatcaaaagttacattgatacagtatttgaattatctaagagt
                          atcaataaagtaccagtagatgtatctgaatctcctggatagtagtaaatagaata
                          cttatacctatgataaatgaagctgaggtatatatgcagatggtgagcaagtaaa
                          gaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccacta
                          gcattaggtgatttaatcggattagatgagattagctataatgaacgattatatact
                          gaataggagatactaaatatagacctcatccactatagctaaaatggttagagct
                          aatcaattaggaagaaaaactaagataggattctatgattataataaataataaga
                          aggagatatacatatgagtacaagtgatgaaaagatatgagaatgtagctgaga
                          agtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgca
                          ataaattcaaagacatagaagaacatatgaagtatagtagatattaataatgatg
                          aaactattgatgagtaatattgacaggggaaggaaaggcatagtagctggagc
                          agatattgcatacatgaaagatttagatgctgtagctgctaaagatatagtatctta
                          ggagcaaaagataggagaaatagaaaatagtaaaaaagtagtgatagctgct
                          gtaaacggatttgattaggtggaggatgtgaacttgcaatggcatgtgatataag
                          aattgcatctgctaaagctaaataggtcagccagaagtaactcaggaataactc
                          caggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagca
                          aaagaattaatattacaggtcaagttataaaagctgatgaagctgaaaaaatagg
                          gctagtaaatagagtcgagagccagacatataatagaagaagagagaaattag
                          ctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata
                          caacaggtgctcaaactgatataaatactggaatagatatagaatctaatttatag
                          gtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaaga
                          gagaagctaacatataaaagggtaataagaaggagatatacatatgagaagatt
                          gaagaagtaattaagtagcaaaagaaagaggacctaaaactatatcagtagcat
                          gagccaagataaagaagattaatggcagagaaatggctagaaaagaaaaaat
                          agcaaatgccatatagtaggagatatagaaaagactaaagaaattgcaaaaag
                          catagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaag
                          catctctaaaatctgagaattagatcacaaggaaaagccgacatggtaatgaaa
                          ggcttagtagacacatcaataatactaaaagcagattaaataaagaagtaggtct
                          tagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgata
                          gattattatcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaa
                          agcaaatcatagaaaatgatgcacagtagcacattcattagatataagtgaacc
                          aaaagagctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatac
                          agagaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggag
                          tatggaggtgggccattgcaattgataatgcagtatattagaagcagctaaaca
                          taaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagata
                          ttgaaggtggtaacatattatataaagctaggtattcactcaaaatcaaaaaatgc
                          aggagttatagaggggctaaagcaccaataatattaacactagagcagacagt
                          gaagaaactaaactaaactcaatagcataggtgattaatggcagcaaaggcata
                          ataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctg
                          gacgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaac
                          ataagacattatcagaagaaataggaaaatatgagaaggtgtctgaccaatag
                          aatacgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacat
                          ctgaattagatgctgtagtaggtagaggaggacacttaaacctataaaaggtggt
                          acttattcagtaagtgctgctatgattgaagatttaaaagtgggagattaggagaa
                          cacgcacaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaat
                          gaccacatacatagtagaccctgagagtagatgaattagaagatgagctagaa
                          tactggtatgcctgaaataagtagagcaagtgtagtacatgattaaatcaaaag
                          gcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaat
                          cttatagagcacacatgggtggaggagtactgaggagctcataaaaatggtaa
                          aatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaaga
                          agtggtggactaccagtaggtgcattagtaaaaatgtgattagtggaaaatatac
                          tcaagatgaaattaaaaagaaaataaaaggtaatggcggactagagcatactta
                          aacactaatgatgctagagaagagaagaaagaattgaagctggtgatgaaaaa
                          gctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctag
                          tgctgcagacttaagggagatgtaaaagcaatattattaactggtggaatcgcat
                          attcaaaaatgatacagaaatgattgcagatagagttaaatttatagcagatgtaa
                          aagatatccaggtgaagatgaaatgattgcattagctcaaggtggacttagagat
                          taactggtgaagaagaggctcaagtttatgataactaa
PfNRS (ribosome           GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT
binding site is           GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC
underlined)               AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC
(SEQ ID NO: 235)          AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC
                          ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA
                          ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG
                          AGATATACAT
Ribosome binding site     CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA
and leader region (SEQ    GATATACAT
ID NO: 236)

Example 18

Assessment of Intestinal Butyrate Levels In Response to SYN501 Administration In Mice

To determine efficacy of butyrate production by the genetically engineered bacteria in vivo, the levels of butyrate upon administration of SYN501 (Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid)) to C57BL6 mice was first assessed in the feces. Water containing 100 mM butyrate was used as a control.

On day 1, C57BL6 mice (24 total animals) were weighed and randomized into 4 groups; Group 1: H2O2O control (n=6); Group 2-100 mM butyrate (n=6); Group 3-streptomycin resistant Nissle (n=6); Group 4-SYN501 (n=6). Mice were either gavaged with 100 ul streptomycin resistant Nissle or SYN501, and group 2 was changed to H20(+)100 mM butyrate at a dose of 10e10 cells/100u1. On days 2-4, mice were weighted and Groups 3 and 4 were gavaged in the AM and the PM with streptomycin resistant Nissle or SYN501. On day 5, mice were weighed and Groups 3 and 4 were gavaged in the am with streptomycin resistant Nissle or SYN501, and feces was collected and butyrate concentrations determined as described in Example 23. Results are depicted in FIG. 10. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM butyrate.

Next the effects of SYN501 on levels of butyrate in the cecum, cecal effluent, large intestine, and large intestine effluent are assessed. Because baseline concentrations of butyrate are high in these compartments, an antibiotic treatment is administered in advance to clear out the bacteria responsible for butyrate production in the intestine. As a result, smaller differences in butyrate levels can be more accurately observed and measured. Water containing 100 mM butyrate is used as a control.

During week 1 of the study, animals are treated with an antibiotic cocktail in the drinking water to reduce the baseline levels of resident microflora. The antibiotic cocktail is composed of ABX-ampicillin, vancomycin, neomycin, and metronidazole. During week 2 animals are orally administered 100 ul of streptomycin resistant Nissle or engineered strain SYN501 twice a day for five days (at a dose of 10e10 cells/100 ul).

On day 1, C57BL6 (Female, 8 weeks) are separated into four groups as follows: Group 1: H20 control (n=10); Group 2: 100 mM butyrate (n=10); Group 3: streptomycin resistant Nissle (n=10); Group 4: SYN501 (n=10). Animals are weighed and feces is collected from the animals (T=0-time point). Animals are changed to H2O (+) antibiotic cocktail. On day 5, animals are weighed and feces is collected (time point T=5d). The H2O (+) antibiotic cocktail bottles are changed. On day 8, the mice are weighed and feces is collected. Mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. The water in all cages is changed to water without antibiotic. Group 2 is provided with 100 mM butyrate in H2O. On days 9-11, mice are weighed, and mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. On day 12, mice are gavaged with streptomycin resistant Nissle or SYN501 in the AM, and 4 hours post dose, blood is harvested, and cecal and large intestinal contents, and tissue, and feces are collected and processed for analysis.

Example 19

Measurement of Satiety Markers Upon Administration of SYN501 In Vivo

To determine whether administration of a butyrate producing strain might result in increased levels of satiety markers, SYN501 is administered to 10-week old C57BL6 (10 weeks) and blood levels of GLP1 and insulin are measured. Butyrate in H20 at 100 mM is used as a control (e.g., as described in Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, PLOS One, April 2012 | Volume 7 ═ Issue 4 ═ e35240).

On day 1, animals are randomized and distributed into 5 groups as follows: Group 1: Time 0 control (n=6); Group 2-H20 (+) 100 mM butyrate, 10 min (n=6); Group 3-SYN501, 30 min (n=6); Group 4-SYN501, 4h (n=6); Group 5-H20 (+) 100 mM butyrate, 4h (n=6). Mice are fasted overnight. On day 2, mice are gavaged with either H20(+)100 mM butyrate or SYN501. Then, blood is harvested via cardiac bleed at the following time points post dose: Group 1 is Time 0; Group 2 (H20 (+) 100 mM butyrate) at 10 min; Group 3 (SYN501) at 30 min; Group 4 (SYN501) at 4 h; Group 5 (H20 (+) 100 mM butyrate) at 4 h. Serum is analyzed by ELISA for GLP-1 and insulin. Fecal samples are analyzed for butyrate by MS as described herein.

Example 20

Comparison of Butyrate Production Levels Between the Genetically Engineered Bacteria Encoding a Butyrate Cassette and Selected Clostridia strains

The efficacy of butyrate production in SYN501 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid) was compared to CBM588 (Clostridia butyricum MIYARISAN, a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain).

Briefly, overnight cultures of SYN501 were diluted 1:100 dilution and was grown in RCM (Reinforced Clostridial Media, which is similar to LB but contains 05% glucose) at 37 C with shaking for 2 hours, then either moved into the anaerobic chamber or left aerobically shaking. Clostridial strains were only grown anaerobically.

At indicated times (2, 8, 24, and 48 h), 120 ul cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 21). Results are depicted in FIG. 12, and show that SYN501 produces butyrate levels comparable to Clostridium spp. in RCM media

Example 21

Quantification of Butyrate by LC-MS/NIS

To obtain the butyrate measurements in Example 37 a LC-MS/MS protocol for butyrate quantification was used.

Sample Preparation

First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 μm/mL sodium butyrate standards were prepared in water. Then, 10 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90 μL of 67% ACN (60 uL ACN+30 uL water per reaction) with 4 ug/mL of butyrate-d7 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 20 μL of diluted samples were added to 180 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour.

LC-MS/MS Method

Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 41 and Table 42. Tandem Mass Spectrometry details are found in Table 43.

TABLE 41
HPLC Details
Column    Thermo Aquasil C18
          column, 5 μm (50 × 2.1 mm)
Mobile    100% H2O, 0.1% Formic
Phase A   Acid
Mobile    100% ACN, 0.1% Formic
Phase B   Acid
Injection 10 uL
volume

TABLE 42
HPLC Method
	Total	Flow
	Time	Rate
	(min)	(μL/min)	A %	B %
	0	0.5	100	0
	1	0.5	100	0
	2	0.5	10	90
	4	0.5	10	90
	4.01	0.5	100	0
	4.25	0.5	100	0

TABLE 43
Tandem Mass Spectrometry Details
Ion Source  HESI-II
Polarity    Positive
SRM         Butyrate 170.0/71.1,
transitions Butyrate d7
            177.1/78.3

Example 22

Quantification of Butyrate in Feces by LC-MS/MS

Sample Preparation

Fresh 1000, 500, 250, 100, 20, 4 and 0.8m/mL sodium butyrate standards were prepared in water. Single fecal pellets were ground in 100 uL water and centrifuged at 15,000 rpm for 5min at 4° C. 10 μL of the sample (fecal supernatant and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90pL of the derivatizing solution containing 50 mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5 ug/mL of butyrate-d₇ were added to each sample. The plate was heat-sealed and incubated at 60° C. for 1 hr. The plate was then centrifuged at 4,000 rpm for 5min and 20 μL of the derivatized samples mixed to 180 μL of 22% acetonitrile with 0.1% formic acid.

LC-MS/MS Method

Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 44 and Table 45. Tandem Mass Spectrometry details are found in Table 46.

TABLE 44
HPLC Details
Column    Luna phenomenex C18
          column, 5 μm (100 × 2.1 mm)
Mobile    100% H2O, 0.1% Formic
Phase A   Acid
Mobile    100% ACN, 0.1% Formic
Phase B   Acid
Injection 10 uL
volume

TABLE 45
HPLC Method
	Total	Flow
	Time	Rate
	(min)	(μL/min)	A %	B %
	0	0.5	95	5
	0.5	0.5	95	5
	1.5	0.5	10	90
	3.5	0.5	10	90
	3.51	0.5	95	5
	3.75	0.5	95	5

TABLE 46
Tandem Mass Spectrometry Details
Ion Source  HESI-II
Polarity    Positive
SRM         Butyrate 230.1/143.1,
transitions Butyrate d7
            237.1/143.1

Example 23

Production of Propionate Through the Sleeping Beauty Mutase Pathway in Genetically Engineered E. coli BW25113 and Nissle

In E. coli , a four gene operon, sbm-ygfD-ygfG-ygfH (sleeping beauty mutase pathway) has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro. While the sleeping beauty mutase pathway is present in E. coli , it is not under the control of a strong promoter and has shown low activity in vivo.

The utility of this operon for the production of propionate was assessed. Because E. coli Nissle does not have the complete operon, initial experiments were conducted in E. coli K12 (BW25113).

First, the native promoter for the sleeping beauty mutase operon on the chromosome in the BW25113 strain was replaced with a fnr promoter (BW25113 ldhA::frt; PfnrS-SBM-cam). The sequence for this construct is provided in Table 47. Mutation of the lactate dehydrogenase gene (ldhA) reportedly increases propionate production, and this mutation is therefore also added in certain embodiments.

TABLE 47
SBM Construct Sequences
Description                     Sequence
BW25113 fnrS SBM
construct (BW25113 frt-
cam-frt-PfnrS-sbm, ygfD,        CCGCCGGG
ygfG, ygfH), comprising rrnB    AGCGGATTTGAACGTTGCGAAGCAACGGC
terminator 1, rrnB terminator 2 CCGGAGGGTGGCGGGCAGGACGCCCGCC
(both italic, uppercase), cat   ATAAACTGCCAGGCATCAAATTAAGC
promoter and cam resistance     TGCGTGG
gene (encoded on the            CCAGTGCCAAGCTTGCATGCAGATTGCAG
lagging strand underlined       CATTACACGTCTTGAGCGATTGTGTAGGCT
uppercase), frt sites (italic   GGAGCTGCTTC
underlined), FNRS promoter      ATTT
bold lowercase, with RBS and    AAATGGCGCGCCTTACGCCCCGCCCTGCC
leader region bold and          A                              CTCATCGCAGTACTGTTGTATTCATTAAG
underlined and FNR binding site CATCTGCCGACATGGAAGCCATCACAAAC
in bold and italics); sleeping  GGCATGATGAACCTGAATCGCCAGCGGCA
beauty operon (sbm, ygfD,       TCAGCACCTTGTCGCCTTGCGTATAATATT
ygfG, ygfH) bold and uppercase  TGCCCATGGTGAAAACGGGGGCGAAGAAG
(SEQ ID NO: 237)                TTGTCCATATTGGCCACGTTTAAATCAAAA
                                CTGGTGAAACTCACCCAGGGATTGGCTGA
                                GACGAAAAACATATTCTCAATAAACCCTTT
                                AGGGAAATAGGCCAGGTTTTCACCGTAAC
                                ACGCCACATCTTGCGAATATATGTGTAGAA
                                ACTGCCGGAAATCGTCGTGGTATTCACTC
                                CAGAGCGATGAAAACGTTTCAGTTTGCTC
                                ATGGAAAACGGTGTAACAAGGGTGAACAC
                                TATCCCATATCACCAGCTCACCGTCTTTCA
                                TTGCCATACGTAATTCCGGATGAGCATTCA
                                TCAGGCGGGCAAGAATGTGAATAAAGGCC
                                GGATAAAACTTGTGCTTATTTTTCTTTACG
                                GTCTTTAAAAAGGCCGTAATATCCAGCTGA
                                ACGGTCTGGTTATAGGTACATTGAGCAAC
                                TGACTGAAATGCCTCAAAATGTTCTTTACG
                                ATGCCATTGGGATATATCAACGGTGGTAT
                                ATCCAGTGATTTTTTTCTCCATTTTAGCTT
                                CCTTAGCTCCTGAAAATCTCGACAACTCAA
                                AAAATACGCCCGGTAGTGATCTTATTTCAT
                                TATGGTGAAAGTTGGAACCTCTTACGTGC
                                CGATCA                            ACGTCTCATTTTCGCCAAAAGTTG
                                GCCCAGGGCTTCCCGGTATCAACAGGGAC
                                ACCAGGATTTATTTATTCTGCGAAGTGATC
                                TTCCGTCACAGGTAGGCGCGCC
                                GGAATA
                                GGAACTAAGGAGGATATTCATATGGACCA
                                TGGCTAATTCCCAGGTACCagttgttcttattggtggt
                                gttgctttatggttgcatcgtagtaaatggttgtaacaaaagcaattttt
                                ccggctgtctgtatacaaaaacgccgcaaagt                            ta
                                aactctctacccattcagggcaatatctctcttggatccaaagtgaa                              ct
                                ctagaaataattttgtttaactttaagaaggagatatacat                            ATGTC
                                TAACGTGCAGGAGTGGCAACAGCTTGCCA
                                ACAAGGAATTGAGCCGTCGGGAGAAAACT
                                GTCGACTCGCTGGTTCATCAAACCGCGGA
                                AGGGATCGCCATCAAGCCGCTGTATACCG
                                AAGCCGATCTCGATAATCTGGAGGTGACA
                                GGTACCCTTCCTGGTTTGCCGCCCTACGTT
                                CGTGGCCCGCGTGCCACTATGTATACCGC
                                CCAACCGTGGACCATCCGTCAGTATGCTG
                                GTTTTTCAACAGCAAAAGAGTCCAACGCTT
                                TTTATCGCCGTAACCTGGCCGCCGGGCAA
                                AAAGGTCTTTCCGTTGCGTTTGACCTTGCC
                                ACCCACCGTGGCTACGACTCCGATAACCC
                                GCGCGTGGCGGGCGACGTCGGCAAAGCG
                                GGCGTCGCTATCGACACCGTGGAAGATAT
                                GAAAGTCCTGTTCGACCAGATCCCGCTGG
                                ATAAAATGTCGGTTTCGATGACCATGAATG
                                GCGCAGTGCTACCAGTACTGGCGTTTTAT
                                ATCGTCGCCGCAGAAGAGCAAGGTGTTAC
                                ACCTGATAAACTGACCGGCACCATTCAAA
                                ACGATATTCTCAAAGAGTACCTCTGCCGCA
                                ACACCTATATTTACCCACCAAAACCGTCAA
                                TGCGCATTATCGCCGACATCATCGCCTGG
                                TGTTCCGGCAACATGCCGCGATTTAATACC
                                ATCAGTATCAGCGGTTACCACATGGGTGA
                                AGCGGGTGCCAACTGCGTGCAGCAGGTAG
                                CATTTACGCTCGCTGATGGGATTGAGTAC
                                ATCAAAGCAGCAATCTCTGCCGGACTGAA
                                AATTGATGACTTCGCTCCTCGCCTGTCGTT
                                CTTCTTCGGCATCGGCATGGATCTGTTTAT
                                GAACGTCGCCATGTTGCGTGCGGCACGTT
                                ATTTATGGAGCGAAGCGGTCAGTGGATTT
                                GGCGCACAGGACCCGAAATCACTGGCGCT
                                GCGTACCCACTGCCAGACCTCAGGCTGGA
                                GCCTGACTGAACAGGATCCGTATAACAAC
                                GTTATCCGCACCACCATTGAAGCGCTGGC
                                TGCGACGCTGGGCGGTACTCAGTCACTGC
                                ATACCAACGCCTTTGACGAAGCGCTTGGT
                                TTGCCTACCGATTTCTCAGCACGCATTGCC
                                CGCAACACCCAGATCATCATCCAGGAAGA
                                ATCAGAACTCTGCCGCACCGTCGATCCAC
                                TGGCCGGATCCTATTACATTGAGTCGCTG
                                ACCGATCAAATCGTCAAACAAGCCAGAGC
                                TATTATCCAACAGATCGACGAAGCCGGTG
                                GCATGGCGAAAGCGATCGAAGCAGGTCTG
                                CCAAAACGAATGATCGAAGAGGCCTCAGC
                                GCGCGAACAGTCGCTGATCGACCAGGGCA
                                AGCGTGTCATCGTTGGTGTCAACAAGTAC
                                AAACTGGATCACGAAGACGAAACCGATGT
                                ACTTGAGATCGACAACGTGATGGTGCGTA
                                ACGAGCAAATTGCTTCGCTGGAACGCATT
                                CGCGCCACCCGTGATGATGCCGCCGTAAC
                                CGCCGCGTTGAACGCCCTGACTCACGCCG
                                CACAGCATAACGAAAACCTGCTGGCTGCC
                                GCTGTTAATGCCGCTCGCGTTCGCGCCAC
                                CCTGGGTGAAATTTCCGATGCGCTGGAAG
                                TCGCTTTCGACCGTTATCTGGTGCCAAGCC
                                AGTGTGTTACCGGCGTGATTGCGCAAAGC
                                TATCATCAGTCTGAGAAATCGGCCTCCGA
                                GTTCGATGCCATTGTTGCGCAAACGGAGC
                                AGTTCCTTGCCGACAATGGTCGTCGCCCG
                                CGCATTCTGATCGCTAAGATGGGCCAGGA
                                TGGACACGATCGCGGCGCGAAAGTGATCG
                                CCAGCGCCTATTCCGATCTCGGTTTCGAC
                                GTAGATTTAAGCCCGATGTTCTCTACACCT
                                GAAGAGATCGCCCGCCTGGCCGTAGAAAA
                                CGACGTTCACGTAGTGGGCGCATCCTCAC
                                TGGCTGCCGGTCATAAAACGCTGATCCCG
                                GAACTGGTCGAAGCGCTGAAAAAATGGGG
                                ACGCGAAGATATCTGCGTGGTCGCGGGTG
                                GCGTCATTCCGCCGCAGGATTACGCCTTC
                                CTGCAAGAGCGCGGCGTGGCGGCGATTTA
                                TGGTCCAGGTACACCTATGCTCGACAGTG
                                TGCGCGACGTACTGAATCTGATAAGCCAG
                                CATCATGATTAATGAAGCCACGCTGGCAG
                                AAAGTATTCGCCGCTTACGTCAGGGTGAG
                                CGTGCCACACTCGCCCAGGCCATGACGCT
                                GGTGGAAAGCCGTCACCCGCGTCATCAGG
                                CACTAAGTACGCAGCTGCTTGATGCCATTA
                                TGCCGTACTGCGGTAACACCCTGCGACTG
                                GGCGTTACCGGCACCCCCGGCGCGGGGAA
                                AAGTACCTTTCTTGAGGCCTTTGGCATGTT
                                GTTGATTCGAGAGGGATTAAAGGTCGCGG
                                TTATTGCGGTCGATCCCAGCAGCCCGGTC
                                ACTGGCGGTAGCATTCTCGGGGATAAAAC
                                CCGCATGAATGACCTGGCGCGTGCCGAAG
                                CGGCGTTTATTCGCCCGGTACCATCCTCC
                                GGTCATCTGGGCGGTGCCAGTCAGCGAGC
                                GCGGGAATTAATGCTGTTATGCGAAGCAG
                                CGGGTTATGACGTAGTGATTGTCGAAACG
                                GTTGGCGTCGGGCAGTCGGAAACAGAAGT
                                CGCCCGCATGGTGGACTGTTTTATCTCGTT
                                GCAAATTGCCGGTGGCGGCGATGATCTGC
                                AGGGCATTAAAAAAGGGCTGATGGAAGTG
                                GCTGATCTGATCGTTATCAACAAAGACGAT
                                GGCGATAACCATACCAATGTCGCCATTGC
                                CCGGCATATGTACGAGAGTGCCCTGCATA
                                TTCTGCGACGTAAATACGACGAATGGCAG
                                CCACGGGTTCTGACTTGTAGCGCACTGGA
                                AAAACGTGGAATCGATGAGATCTGGCACG
                                CCATCATCGACTTCAAAACCGCGCTAACTG
                                CCAGTGGTCGTTTACAACAAGTGCGGCAA
                                CAACAATCGGTGGAATGGCTGCGTAAGCA
                                GACCGAAGAAGAAGTACTGAATCACCTGT
                                TCGCGAATGAAGATTTCGATCGCTATTACC
                                GCCAGACGCTTTTAGCGGTCAAAAACAAT
                                ACGCTCTCACCGCGCACCGGCCTGCGGCA
                                GCTCAGTGAATTTATCCAGACGCAATATTT
                                TGATTAAAGGAATTTTTATGTCTTATCAGTA
                                TGTTAACGTTGTCACTATCAACAAAGTGGC
                                GGTCATTGAGTTTAACTATGGCCGAAAACT
                                TAATGCCTTAAGTAAAGTCTTTATTGATGA
                                TCTTATGCAGGCGTTAAGCGATCTCAACC
                                GGCCGGAAATTCGCTGTATCATTTTGCGC
                                GCACCGAGTGGATCCAAAGTCTTCTCCGC
                                AGGTCACGATATTCACGAACTGCCGTCTG
                                GCGGTCGCGATCCGCTCTCCTATGATGAT
                                CCATTGCGTCAAATCACCCGCATGATCCAA
                                AAATTCCCGAAACCGATCATTTCGATGGTG
                                GAAGGTAGTGTTTGGGGTGGCGCATTTGA
                                AATGATCATGAGTTCCGATCTGATCATCGC
                                CGCCAGTACCTCAACCTTCTCAATGACGCC
                                TGTAAACCTCGGCGTCCCGTATAACCTGG
                                TCGGCATTCACAACCTGACCCGCGACGCG
                                GGCTTCCACATTGTCAAAGAGCTGATTTTT
                                ACCGCTTCGCCAATCACCGCCCAGCGCGC
                                GCTGGCTGTCGGCATCCTCAACCATGTTG
                                TGGAAGTGGAAGAACTGGAAGATTTCACC
                                TTACAAATGGCGCACCACATCTCTGAGAA
                                AGCGCCGTTAGCCATTGCCGTTATCAAAG
                                AAGAGCTGCGTGTACTGGGCGAAGCACAC
                                ACCATGAACTCCGATGAATTTGAACGTATT
                                CAGGGGATGCGCCGCGCGGTGTATGACAG
                                CGAAGATTACCAGGAAGGGATGAACGCTT
                                TCCTCGAAAAACGTAAACCTAATTTCGTTG
                                GTCATTAATCCCTGCGAACGAAGGAGTAAA
                                AATGGAAACTCAGTGGACAAGGATGACCG
                                CCAATGAAGCGGCAGAAATTATCCAGCAT
                                AACGACATGGTGGCATTTAGCGGCTTTAC
                                CCCGGCGGGTTCGCCGAAAGCCCTACCCA
                                CCGCGATTGCCCGCAGAGCTAACGAACAG
                                CATGAGGCCAAAAAGCCGTATCAAATTCG
                                CCTTCTGACGGGTGCGTCAATCAGCGCCG
                                CCGCTGACGATGTACTTTCTGACGCCGAT
                                GCTGTTTCCTGGCGTGCGCCATATCAAAC
                                ATCGTCCGGTTTACGTAAAAAGATCAATCA
                                GGGCGCGGTGAGTTTCGTTGACCTGCATT
                                TGAGCGAAGTGGCGCAAATGGTCAATTAC
                                GGTTTCTTCGGCGACATTGATGTTGCCGTC
                                ATTGAAGCATCGGCACTGGCACCGGATGG
                                TCGAGTCTGGTTAACCAGCGGGATCGGTA
                                ATGCGCCGACCTGGCTGCTGCGGGCGAAG
                                AAAGTGATCATTGAACTCAATCACTATCAC
                                GATCCGCGCGTTGCAGAACTGGCGGATAT
                                TGTGATTCCTGGCGCGCCACCGCGGCGCA
                                ATAGCGTGTCGATCTTCCATGCAATGGATC
                                GCGTCGGTACCCGCTATGTGCAAATCGAT
                                CCGAAAAAGATTGTCGCCGTCGTGGAAAC
                                CAACTTGCCCGACGCCGGTAATATGCTGG
                                ATAAGCAAAATCCCATGTGCCAGCAGATT
                                GCCGATAACGTGGTCACGTTCTTATTGCA
                                GGAAATGGCGCATGGGCGTATTCCGCCGG
                                AATTTCTGCCGCTGCAAAGTGGCGTGGGC
                                AATATCAATAATGCGGTAATGGCGCGTCT
                                GGGGGAAAACCCGGTAATTCCTCCGTTTA
                                TGATGTATTCGGAAGTGCTACAGGAATCG
                                GTGGTGCATTTACTGGAAACCGGCAAAAT
                                CAGCGGGGCCAGCGCCTCCAGCCTGACAA
                                TCTCGGCCGATTCCCTGCGCAAGATTTAC
                                GACAATATGGATTACTTTGCCAGCCGCATT
                                GTGTTGCGTCCGCAGGAGATTTCCAATAA
                                CCCGGAAATCATCCGTCGTCTGGGCGTCA
                                TCGCTCTGAACGTCGGCCTGGAGTTTGAT
                                ATTTACGGGCATGCCAACTCAACACACGT
                                AGCCGGGGTCGATCTGATGAACGGCATCG
                                GCGGCAGCGGTGATTTTGAACGCAACGCG
                                TATCTGTCGATCTTTATGGCCCCGTCGATT
                                GCTAAAGAAGGCAAGATCTCAACCGTCGT
                                GCCAATGTGCAGCCATGTTGATCACAGCG
                                AACACAGCGTCAAAGTGATCATCACCGAA
                                CAAGGGATCGCCGATCTGCGCGGTCTTTC
                                CCCGCTTCAACGCGCCCGCACTATCATTG
                                ATAATTGTGCACATCCTATGTATCGGGATT
                                ATCTGCATCGCTATCTGGAAAATGCGCCT
                                GGCGGACATATTCACCACGATCTTAGCCA
                                CGTCTTCGACTTACACCGTAATTTAATTGC
                                AACCGGCTCGATGCTGGGTTAA
FNRS promoter bold lowercase,   agttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggtt
with RBS and leader region      gtaacaaaagcaatttttccggctgtctgtatacaaaaacgccgcaaa
bold and underlined, and FNR    gt                            taaactctctacccattcagggcaatatctctct
binding site bold and italics); tggatccaaagtgaa                              ctctagaaataattttgtttaactttaagaagga
sleeping beauty operon (sbm,    gatatacat                            ATGTCTAACGTGCAGGAGTGGCAA
ygfD, ygfG, ygfH) bold and      CAGCTTGCCAACAAGGAATTGAGCCGTCG
uppercase                       GGAGAAAACTGTCGACTCGCTGGTTCATC
(SEQ ID NO: 238)                AAACCGCGGAAGGGATCGCCATCAAGCCG
                                CTGTATACCGAAGCCGATCTCGATAATCTG
                                GAGGTGACAGGTACCCTTCCTGGTTTGCC
                                GCCCTACGTTCGTGGCCCGCGTGCCACTA
                                TGTATACCGCCCAACCGTGGACCATCCGT
                                CAGTATGCTGGTTTTTCAACAGCAAAAGA
                                GTCCAACGCTTTTTATCGCCGTAACCTGGC
                                CGCCGGGCAAAAAGGTCTTTCCGTTGCGT
                                TTGACCTTGCCACCCACCGTGGCTACGAC
                                TCCGATAACCCGCGCGTGGCGGGCGACGT
                                CGGCAAAGCGGGCGTCGCTATCGACACCG
                                TGGAAGATATGAAAGTCCTGTTCGACCAG
                                ATCCCGCTGGATAAAATGTCGGTTTCGAT
                                GACCATGAATGGCGCAGTGCTACCAGTAC
                                TGGCGTTTTATATCGTCGCCGCAGAAGAG
                                CAAGGTGTTACACCTGATAAACTGACCGG
                                CACCATTCAAAACGATATTCTCAAAGAGTA
                                CCTCTGCCGCAACACCTATATTTACCCACC
                                AAAACCGTCAATGCGCATTATCGCCGACA
                                TCATCGCCTGGTGTTCCGGCAACATGCCG
                                CGATTTAATACCATCAGTATCAGCGGTTAC
                                CACATGGGTGAAGCGGGTGCCAACTGCGT
                                GCAGCAGGTAGCATTTACGCTCGCTGATG
                                GGATTGAGTACATCAAAGCAGCAATCTCT
                                GCCGGACTGAAAATTGATGACTTCGCTCC
                                TCGCCTGTCGTTCTTCTTCGGCATCGGCAT
                                GGATCTGTTTATGAACGTCGCCATGTTGC
                                GTGCGGCACGTTATTTATGGAGCGAAGCG
                                GTCAGTGGATTTGGCGCACAGGACCCGAA
                                ATCACTGGCGCTGCGTACCCACTGCCAGA
                                CCTCAGGCTGGAGCCTGACTGAACAGGAT
                                CCGTATAACAACGTTATCCGCACCACCATT
                                GAAGCGCTGGCTGCGACGCTGGGCGGTAC
                                TCAGTCACTGCATACCAACGCCTTTGACGA
                                AGCGCTTGGTTTGCCTACCGATTTCTCAGC
                                ACGCATTGCCCGCAACACCCAGATCATCA
                                TCCAGGAAGAATCAGAACTCTGCCGCACC
                                GTCGATCCACTGGCCGGATCCTATTACATT
                                GAGTCGCTGACCGATCAAATCGTCAAACA
                                AGCCAGAGCTATTATCCAACAGATCGACG
                                AAGCCGGTGGCATGGCGAAAGCGATCGAA
                                GCAGGTCTGCCAAAACGAATGATCGAAGA
                                GGCCTCAGCGCGCGAACAGTCGCTGATCG
                                ACCAGGGCAAGCGTGTCATCGTTGGTGTC
                                AACAAGTACAAACTGGATCACGAAGACGA
                                AACCGATGTACTTGAGATCGACAACGTGA
                                TGGTGCGTAACGAGCAAATTGCTTCGCTG
                                GAACGCATTCGCGCCACCCGTGATGATGC
                                CGCCGTAACCGCCGCGTTGAACGCCCTGA
                                CTCACGCCGCACAGCATAACGAAAACCTG
                                CTGGCTGCCGCTGTTAATGCCGCTCGCGT
                                TCGCGCCACCCTGGGTGAAATTTCCGATG
                                CGCTGGAAGTCGCTTTCGACCGTTATCTG
                                GTGCCAAGCCAGTGTGTTACCGGCGTGAT
                                TGCGCAAAGCTATCATCAGTCTGAGAAAT
                                CGGCCTCCGAGTTCGATGCCATTGTTGCG
                                CAAACGGAGCAGTTCCTTGCCGACAATGG
                                TCGTCGCCCGCGCATTCTGATCGCTAAGA
                                TGGGCCAGGATGGACACGATCGCGGCGCG
                                AAAGTGATCGCCAGCGCCTATTCCGATCT
                                CGGTTTCGACGTAGATTTAAGCCCGATGTT
                                CTCTACACCTGAAGAGATCGCCCGCCTGG
                                CCGTAGAAAACGACGTTCACGTAGTGGGC
                                GCATCCTCACTGGCTGCCGGTCATAAAAC
                                GCTGATCCCGGAACTGGTCGAAGCGCTGA
                                AAAAATGGGGACGCGAAGATATCTGCGTG
                                GTCGCGGGTGGCGTCATTCCGCCGCAGGA
                                TTACGCCTTCCTGCAAGAGCGCGGCGTGG
                                CGGCGATTTATGGTCCAGGTACACCTATG
                                CTCGACAGTGTGCGCGACGTACTGAATCT
                                GATAAGCCAGCATCATGATTAATGAAGCC
                                ACGCTGGCAGAAAGTATTCGCCGCTTACG
                                TCAGGGTGAGCGTGCCACACTCGCCCAGG
                                CCATGACGCTGGTGGAAAGCCGTCACCCG
                                CGTCATCAGGCACTAAGTACGCAGCTGCT
                                TGATGCCATTATGCCGTACTGCGGTAACA
                                CCCTGCGACTGGGCGTTACCGGCACCCCC
                                GGCGCGGGGAAAAGTACCTTTCTTGAGGC
                                CTTTGGCATGTTGTTGATTCGAGAGGGATT
                                AAAGGTCGCGGTTATTGCGGTCGATCCCA
                                GCAGCCCGGTCACTGGCGGTAGCATTCTC
                                GGGGATAAAACCCGCATGAATGACCTGGC
                                GCGTGCCGAAGCGGCGTTTATTCGCCCGG
                                TACCATCCTCCGGTCATCTGGGCGGTGCC
                                AGTCAGCGAGCGCGGGAATTAATGCTGTT
                                ATGCGAAGCAGCGGGTTATGACGTAGTGA
                                TTGTCGAAACGGTTGGCGTCGGGCAGTCG
                                GAAACAGAAGTCGCCCGCATGGTGGACTG
                                TTTTATCTCGTTGCAAATTGCCGGTGGCGG
                                CGATGATCTGCAGGGCATTAAAAAAGGGC
                                TGATGGAAGTGGCTGATCTGATCGTTATC
                                AACAAAGACGATGGCGATAACCATACCAA
                                TGTCGCCATTGCCCGGCATATGTACGAGA
                                GTGCCCTGCATATTCTGCGACGTAAATAC
                                GACGAATGGCAGCCACGGGTTCTGACTTG
                                TAGCGCACTGGAAAAACGTGGAATCGATG
                                AGATCTGGCACGCCATCATCGACTTCAAA
                                ACCGCGCTAACTGCCAGTGGTCGTTTACA
                                ACAAGTGCGGCAACAACAATCGGTGGAAT
                                GGCTGCGTAAGCAGACCGAAGAAGAAGTA
                                CTGAATCACCTGTTCGCGAATGAAGATTTC
                                GATCGCTATTACCGCCAGACGCTTTTAGC
                                GGTCAAAAACAATACGCTCTCACCGCGCA
                                CCGGCCTGCGGCAGCTCAGTGAATTTATC
                                CAGACGCAATATTTTGATTAAAGGAATTTT
                                TATGTCTTATCAGTATGTTAACGTTGTCAC
                                TATCAACAAAGTGGCGGTCATTGAGTTTAA
                                CTATGGCCGAAAACTTAATGCCTTAAGTAA
                                AGTCTTTATTGATGATCTTATGCAGGCGTT
                                AAGCGATCTCAACCGGCCGGAAATTCGCT
                                GTATCATTTTGCGCGCACCGAGTGGATCC
                                AAAGTCTTCTCCGCAGGTCACGATATTCAC
                                GAACTGCCGTCTGGCGGTCGCGATCCGCT
                                CTCCTATGATGATCCATTGCGTCAAATCAC
                                CCGCATGATCCAAAAATTCCCGAAACCGA
                                TCATTTCGATGGTGGAAGGTAGTGTTTGG
                                GGTGGCGCATTTGAAATGATCATGAGTTC
                                CGATCTGATCATCGCCGCCAGTACCTCAA
                                CCTTCTCAATGACGCCTGTAAACCTCGGC
                                GTCCCGTATAACCTGGTCGGCATTCACAA
                                CCTGACCCGCGACGCGGGCTTCCACATTG
                                TCAAAGAGCTGATTTTTACCGCTTCGCCAA
                                TCACCGCCCAGCGCGCGCTGGCTGTCGGC
                                ATCCTCAACCATGTTGTGGAAGTGGAAGA
                                ACTGGAAGATTTCACCTTACAAATGGCGC
                                ACCACATCTCTGAGAAAGCGCCGTTAGCC
                                ATTGCCGTTATCAAAGAAGAGCTGCGTGT
                                ACTGGGCGAAGCACACACCATGAACTCCG
                                ATGAATTTGAACGTATTCAGGGGATGCGC
                                CGCGCGGTGTATGACAGCGAAGATTACCA
                                GGAAGGGATGAACGCTTTCCTCGAAAAAC
                                GTAAACCTAATTTCGTTGGTCATTAATCCC
                                TGCGAACGAAGGAGTAAAAATGGAAACTCA
                                GTGGACAAGGATGACCGCCAATGAAGCGG
                                CAGAAATTATCCAGCATAACGACATGGTG
                                GCATTTAGCGGCTTTACCCCGGCGGGTTC
                                GCCGAAAGCCCTACCCACCGCGATTGCCC
                                GCAGAGCTAACGAACAGCATGAGGCCAAA
                                AAGCCGTATCAAATTCGCCTTCTGACGGG
                                TGCGTCAATCAGCGCCGCCGCTGACGATG
                                TACTTTCTGACGCCGATGCTGTTTCCTGGC
                                GTGCGCCATATCAAACATCGTCCGGTTTAC
                                GTAAAAAGATCAATCAGGGCGCGGTGAGT
                                TTCGTTGACCTGCATTTGAGCGAAGTGGC
                                GCAAATGGTCAATTACGGTTTCTTCGGCG
                                ACATTGATGTTGCCGTCATTGAAGCATCG
                                GCACTGGCACCGGATGGTCGAGTCTGGTT
                                AACCAGCGGGATCGGTAATGCGCCGACCT
                                GGCTGCTGCGGGCGAAGAAAGTGATCATT
                                GAACTCAATCACTATCACGATCCGCGCGTT
                                GCAGAACTGGCGGATATTGTGATTCCTGG
                                CGCGCCACCGCGGCGCAATAGCGTGTCGA
                                TCTTCCATGCAATGGATCGCGTCGGTACC
                                CGCTATGTGCAAATCGATCCGAAAAAGAT
                                TGTCGCCGTCGTGGAAACCAACTTGCCCG
                                ACGCCGGTAATATGCTGGATAAGCAAAAT
                                CCCATGTGCCAGCAGATTGCCGATAACGT
                                GGTCACGTTCTTATTGCAGGAAATGGCGC
                                ATGGGCGTATTCCGCCGGAATTTCTGCCG
                                CTGCAAAGTGGCGTGGGCAATATCAATAA
                                TGCGGTAATGGCGCGTCTGGGGGAAAACC
                                CGGTAATTCCTCCGTTTATGATGTATTCGG
                                AAGTGCTACAGGAATCGGTGGTGCATTTA
                                CTGGAAACCGGCAAAATCAGCGGGGCCAG
                                CGCCTCCAGCCTGACAATCTCGGCCGATT
                                CCCTGCGCAAGATTTACGACAATATGGATT
                                ACTTTGCCAGCCGCATTGTGTTGCGTCCG
                                CAGGAGATTTCCAATAACCCGGAAATCAT
                                CCGTCGTCTGGGCGTCATCGCTCTGAACG
                                TCGGCCTGGAGTTTGATATTTACGGGCAT
                                GCCAACTCAACACACGTAGCCGGGGTCGA
                                TCTGATGAACGGCATCGGCGGCAGCGGTG
                                ATTTTGAACGCAACGCGTATCTGTCGATCT
                                TTATGGCCCCGTCGATTGCTAAAGAAGGC
                                AAGATCTCAACCGTCGTGCCAATGTGCAG
                                CCATGTTGATCACAGCGAACACAGCGTCA
                                AAGTGATCATCACCGAACAAGGGATCGCC
                                GATCTGCGCGGTCTTTCCCCGCTTCAACG
                                CGCCCGCACTATCATTGATAATTGTGCACA
                                TCCTATGTATCGGGATTATCTGCATCGCTA
                                TCTGGAAAATGCGCCTGGCGGACATATTC
                                ACCACGATCTTAGCCACGTCTTCGACTTAC
                                ACCGTAATTTAATTGCAACCGGCTCGATGC
                                TGGGTTAA

Next, this strain was tested for propionate production.

Briefly, 3 ml LB (containing selective antibiotics (cam) where necessary was inoculated from frozen glycerol stocks with either wild type E. coli K12 or the genetically engineered bacteria comprising the chromosomal sleeping beauty mutase operon under the control of a FNR promoter. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, and 24 hours, 120 ul of cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for propionate concentrations, as described in

Results are depicted in FIG. 22B and show that the genetically engineered strain produces ˜2.5 mM after 24 h, while very little or no propionate production was detected from the E. coli K12 wild type strain. Propionate was measured as described in Example 26.

Example 24

Evaluation of the Sleeping Beauty Mutase Pathway for the Production of Propionate in E coli Nissle

Next, the SBM pathway is evaluated for propionate production in E. coli Nissle. Nissle does not have the full 4-gene sleeping beauty mutase operon; it only has the first gene and a partial gene of the second, and genes 3 and 4 are missing. Therefore, recombineering is used to introduce this pathway into Nissle. The frt-cam-frt-PfnrS-sbm, ygfD, ygfG, ygfH construct is inserted at the location of the endogenous, truncated Nissle SBM. Next, the construct is transformed into E coli Nissle and tested for propionate production essentially as described above.

Example 25

Evaluation of the Acrylate Pathway from Clostridium propionicum for Propionate Production

The acrylate pathway from Clostridium propionicum is evaluated for adaptation to propionate production in E. coli . A construct (Ptet-pct-1cdABC-acrABC), codon optimized for E. coli , was synthesized by Genewiz and placed in a high copy plasmid (Logic051). Additionally, another construct is generated for side by side testing, in which the acrABC genes (which may be the rate limiting step of the pathway) are replaced with the acuI gene from Rhodobacter sphaeroides (Ptet- acuI-pct-lcdABC). Subsequently these constructs are transformed into BW25113 and are assessed for their ability to produce propionate, as compared to the type BW5113 strain as described above in Example 23. Propionate was measured as described in Example 26.

Table 48 of Exemplary Propionate Cassette Sequences
Description
and SEQ ID
NO             Sequence
Ptet-pct-      ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataaga
lcdABC-        aggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactat
acrABC; Ptet:  cagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaa
lower case;    gtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataa
tertR/tetA     aaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacctaaatgtacttttgc
promoter       tccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgcca
within Ptet:   gctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgt
lower case     cgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg
bold, with tet cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatca
operator:      ctttacttttatctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagtt
lower case     attttaccac                              tccctatcagtgatagag                            aaaagtgaactctagaaataattttgtttaacttta
bold           a                              gaaggagatatacat              ATGCGCAAAGTGCCGATTATCACGGCTG
underlined;    ACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTG
ribosome       ACAACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCC
binding site   CTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAACGGGC
and leader:    GAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAG
lowe case      GGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGCAT
italic;        GAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCG
ribosome       ACCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAA
binding sites: AATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCA
lower case     TCTCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTC
underlined;    ACGAAAGTAGGAATCGGCACCTTCATTGATCCACGTAAC
coding         GGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATAT
regions: upper CGTAGAACTGGTGGAAATTAAAGGGCAGGAATACCTGTT
case; (SEQ ID  TTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGC
NO: 239)       ACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAAA
               GAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCG
               GTGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAG
               CGTGTCGTCAAAGCAGGCACGCTGGATCCGCGCCATGTG
               AAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCG
               GATCCGGAGGACCATCAACAGTCCCTTGACTGCGAATAT
               GATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTG
               GTGGGTGAACCACTGCCTTTATCCGCGAAGAAAGTCATC
               GGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCA
               GTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCC
               GTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTC
               ACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGC
               GGTGTTCGCTTTGGGGCGAGCTACAATGCTGACGCCTTG
               ATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGG
               GGTCTGGATCTTTGTTACCTGGGTTTAGCTGAATGCGACG
               AAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTA
               TCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACA
               CGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGCCGGGG
               GGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCG
               TTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTG
               GAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAAC
               AAGCAACAAGTTACCTACATCACGGAGCGTTGCGTCTTC
               CTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCG
               CCAGGCATCGATCTGCAGACCCAGATTTTGGATGTTATG
               GACTTTGCCCCGATCATTGATCGTGACGCAAACGGGCAG
               ATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTG
               ATGGGCTTGAAAGAAATGAAGTCTTGAtaagaaggagatatacatA
               TGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAG
               CATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAG
               CGAAAGCCCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAG
               TCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCA
               TGATCTACCCGGAGACTCATGCAGCGGGCATCGGTGCCC
               GCAAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGC
               AAAGGCTACAACGTGGATTGTTGTTCCTACGGCCGTGTA
               AATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATC
               ACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCT
               GCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATA
               ATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAG
               CAGCAGAACTCGATATTCCTTGCATCGTGATCGACGTAC
               CGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGG
               CCTACATCGCGGACCAGTTCCGCAATGCAATTTCTCAGC
               TGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAAT
               TTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACC
               ACTGGAACCGCATTGCCGAGATGGCGAAATACAAGCCTA
               GCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTT
               AATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTAC
               CTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAA
               GGCGGGTATCTACGCCTTTAAAGGTGCGGAAAAAACGCG
               CTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGG
               TCACACGTTTAAATCTATGAAGAATCTGAATTCGATTAT
               GACCGGTACGGCATACCCCGCCCTTTGGGACCTGCACTA
               TGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGC
               GTACACCCGTATTTATATTAATACTTGTCTGCAGAACAA
               AGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGT
               GGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACT
               GATGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAA
               AGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGA
               TCAGACCGATCCTCGCGTTTTTTCTCCGGCCCAGTTTGAT
               ACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAAT
               ATGGCGGCAGCGGAATAAtaagaaggagatatacatATGTCACGC
               GTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCG
               AATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAAC
               AGGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCC
               CGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGG
               AATCTGGGGCGCCCAGGGCAAAACGATTAGTAAAGCGC
               GCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCA
               GGTTATGGAATTACAGTGCGAGGGCGCGTATGATGACCT
               GTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCAAA
               TGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATT
               GTATTTACGCATCCGCAGAACCGCGGATTAGAAGCGGCG
               AACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCA
               CAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCC
               GCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGT
               GCCGTGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTAT
               CCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTT
               AAAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCA
               CTTGTGAAAGAACTGATCGCTGAGATTAAAGCAACGCCA
               GTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGG
               CATTCTGTTGGAACCGAATGAGTTATTAGATATCTTTAAT
               GAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAG
               GAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAA
               GGCGGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAA
               ATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGT
               CGCGGCCGTATGTTAATTAACAAAACGATTCAGACCGGT
               GCGGACGCTATCGTAGTTGCAATGATGAAGTTTTGCGAC
               CCAGAAGAATGGGATTATCCGGTAATGTACCGTGAATTT
               GAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGA
               TCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCT
               GCAGTCATTCGTCGAAATGCTTTAAtaagaaggagatatacatATG
               TATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAG
               CGGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCG
               AGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAAC
               GCGCACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAA
               AGGAAGACATCAGCTACACAGTAGCTACGGGCTATGGG
               CGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAA
               ATTAGCTGTCATGCCAAAGGCATTTATTTCTTAGTACCAA
               CTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGA
               AAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGCAA
               TTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGT
               TTCCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTG
               GATGAAATGGCTGAACTGGATGAACAGGCGACTGACAC
               CGCTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAG
               CGAAGTAATTAGCCAATTGAGCAATGGTGTCTCACGCAA
               CAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACG
               TGCGTGTGGTCTGGCGTATCGCGGCGGTTTGGAGAAAGA
               TGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGT
               GGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTAT
               CGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGC
               AGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAAtaa
               gaaggagatatacatATGGCCTTCAATAGCGCAGATATTAATTCT
               TTCCGCGATATTTGGGTGTTTTGTGAACAGCGTGAGGGC
               AAACTGATTAACACCGATTTCGAATTAATTAGCGAAGGT
               CGTAAACTGGCTGACGAACGCGGAAGCAAACTGGTTGG
               AATTTTGCTGGGGCACGAAGTTGAAGAAATCGCAAAAG
               AATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGTGCG
               ATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGC
               CAAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGA
               GGTAATTTTGATCGGTGCCACCAACATTGGCCGTGATCT
               CGGACCGCGTTGTGCTGCACGCTTGCACACGGGGCTGAC
               GGCTGATTGCACGCACCTGGATATTGATATGAATAAATA
               TGTGGACTTTCTTAGCACCAGTAGCACCTTGGATATCTCG
               TCGATGACTTTCCCTATGGAAGATACAAACCTTAAAATG
               ACGCGCCCTGCATTTGGCGGACATCTGATGGCAACGATC
               ATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGTGCGCC
               CCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATG
               GCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTG
               TCGGATGAAGACCTTAAAACTAAAGTAATTAATATCGTG
               AAGGAAACGAAAAAGATTGTGGATCTGATCGGCGCAGA
               AATTATTGTGTCAGTTGGTCGTGGTATCTCGAAAGATGTC
               CAAGGTGGAATTGCACTGGCTGAAAAACTTGCGGACGCA
               TTTGGTAACGGTGTCGTGGGCGGCTCGCGCGCAGTGATT
               GATTCCGGCTGGTTACCTGCGGATCATCAGGTTGGACAA
               ACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCGCTG
               GGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAA
               GACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACG
               GCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGT
               GATTTATTTAAAATCGTACCGATGATGATCGACGCGATC
               AAAGAGGGTAAAAACGCATGAtaagaaggagatatacatATGCGC
               ATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGGC
               AAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCC
               TCAATGGCAGCGATTATTAACCCGGACGATATGTCCGCG
               ATCGAACAGGCATTAAAACTGAAAGATGAAACCGGATG
               CCAGGTTACGGCGCTTACGATGGGTCCTCCTCCTGCCGA
               GGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGACGA
               TGGTGTGCTGATTTCGGCCCGTGAATTTGGGGGGTCCGA
               TACCTTCGCAACCAGTCAAATTATTAGCGCGGCAATCCA
               TAAATTAGGCTTAAGCAATGAAGACATGATCTTTTGCGG
               TCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCC
               TCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTA
               TGGCGCAGGAATCAAAAAATCTGGTGATTTAGTGCTGGT
               GAAGCGTATGTTGGAGGATGGTTATATGATGATCGAAGT
               CGAAACTCCATGTCTGATTACCTGCATTCAGGATAAAGC
               GGTAAAACCACGTTACATGACTCTCAACGGTATTATGGA
               ATGCTACTCCAAGCCGCTCCTCGTTCTCGATTACGAAGC
               ACTGAAAGATGAACCGCTGATCGAACTTGATACCATTGG
               GCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTTTAC
               GCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCAC
               CGATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGA
               TGCAGAAACATGTCATCTAAtaagaaggagatatacatATGTTCTT
               ACTGAAGATTAAAAAAGAACGTATGAAACGCATGGACT
               TTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGG
               CGCGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCG
               AAGAGATTGATCGTGAGCACGTTTTTCCTGCAGAAAACT
               TTAAGAAGATGGCGGAAATTGGCTTAACCGGCATTGGTA
               TCCCGAAAGAATTTGGTGGCTCCGGTGGAGGCACCCTGG
               AGAAGGTCATTGCCGTGTCAGAATTCGGCAAAAAGTGTA
               TGGCCTCAGCTTCCATTTTAAGCATTCATCTTATCGCGCC
               GCAGGCAATCTACAAATATGGGACCAAAGAACAGAAAG
               AGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAACTGG
               GCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATG
               CCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAG
               ACAAACGAGTACGTGCTGAATGGCACCAAATGCTTTATC
               AGCGGGGGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCG
               CTTACTGAACCGAAAAAAGGTCTGAAAGGGATGAGCGC
               GATTATCGTGGAGAAAGGGACCCCGGGCTTCAGCATCGG
               CAAGGTGGAGAGCAAGATGGGGATCGCAGGTTCGGAAA
               CCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGCTG
               CCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTG
               CTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCG
               CTCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGA
               GTGTGAAGTACGTTCACGAGCGCATTCAATTTGGTAAAC
               CGATCGCGAATCTGCAGGGAATTCAATGGTATATCGCGG
               ATATGGCGACCAAAACCGCCGCGGCACGCGCACTTGTTG
               AGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCGTTCA
               CAAAGGAATCTGCTATGTGCAAGCTGAACGCCTCCGAAA
               ACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCACG
               GGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTA
               TGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGA
               CATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAA
               TGAAACGCTAA
pct-lcdABC-    ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCA
acrABC         AAACTGATCAAGGACGGCGACACCGTGACAACTAGCGG
(ribosome      CTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGC
binding sites: AGTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGA
lower case     ACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCG
underlined;    ACGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGC
coding         TGAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGG
regions: upper CGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCC
case) (SEQ ID  TACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCG
NO: 240)       ATATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAG
               GAATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGA
               AGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGG
               TGGAAATTAAAGGGCAGGAATACCTGTTTTATCCGGCGT
               TCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGG
               ACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCG
               CCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAAC
               TCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC
               AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCG
               GGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAG
               GACCATCAACAGTCCCTTGACTGCGAATATGATCCTGCC
               CTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAA
               CCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGT
               GGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTT
               GGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGAT
               GAAGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAG
               TCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGC
               TTTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAG
               GGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGAT
               CTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGT
               AATATCAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGT
               GCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAG
               TCTTCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAG
               TGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAG
               GGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATC
               ACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAA
               GTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAG
               AAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCG
               ATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCC
               GATCATTGATCGTGACGCAAACGGGCAGATTAAACTGAT
               GGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAA
               AGAAATGAAGTCTTGAtaagaaggagatatacatATGAGCTTAACC
               CAAGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAG
               GGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCG
               CGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCC
               GGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCC
               GGAGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTG
               CGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTAC
               AACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGT
               TACATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTC
               AAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTC
               CGCTTCCCGATTTGGTGATTACGTGTAATAATATCTGTAA
               CACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT
               CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCAT
               ACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCG
               GACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTT
               GTGGCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCA
               AAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCA
               TTGCCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACG
               GCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTG
               CCGCAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTT
               CGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCT
               ACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGG
               AAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTA
               AATCTATGAAGAATCTGAATTCGATTATGACCGGTACGG
               CATACCCCGCCCTTTGGGACCTGCACTATGACGCTAACG
               ACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTA
               TTTATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCC
               TGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCG
               TATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCT
               GAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACG
               GTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCC
               TCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAG
               GCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCG
               GAATAAtaagaaggagatatacatATGTCACGCGTGGAGGCAATCC
               TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA
               GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC
               GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT
               ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC
               CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC
               TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT
               ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT
               TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG
               AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT
               CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG
               GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA
               GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT
               TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT
               GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT
               GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG
               TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA
               CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG
               GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA
               CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC
               GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCAG
               ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC
               TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC
               TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG
               TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC
               GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG
               GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG
               GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG
               TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG
               AAATGCTTTAAtaagaaggagatatacatATGTATACCTTGGGGAT
               TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA
               AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT
               CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA
               AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG
               CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG
               CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC
               CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT
               ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG
               GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT
               AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG
               GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA
               CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC
               ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA
               TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT
               GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG
               TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT
               GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC
               GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC
               TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA
               TGAGGCCGCCCAGAAGAAGTAAtaagaaggagatatacatATGGC
               CTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGG
               GTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACC
               GATTTCGAATTAATTAGCGAAGGTCGTAAACTGGCTGAC
               GAACGCGGAAGCAAACTGGTTGGAATTTTGCTGGGGCAC
               GAAGTTGAAGAAATCGCAAAAGAATTAGGCGGCTATGG
               TGCGGACAAGGTAATTGTGTGCGATCATCCGGAACTTAA
               ATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGAC
               GTCGTGATGGAAGAGAAACCGGAGGTAATTTTGATCGGT
               GCCACCAACATTGGCCGTGATCTCGGACCGCGTTGTGCT
               GCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCAC
               CTGGATATTGATATGAATAAATATGTGGACTTTCTTAGC
               ACCAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTA
               TGGAAGATACAAACCTTAAAATGACGCGCCCTGCATTTG
               GCGGACATCTGATGGCAACGATCATTTGTCCACGCTTCC
               GTCCCTGTATGAGCACAGTGCGCCCCGGAGTGATGAAGA
               AAGCGGAGTTCTCGCAGGAGATGGCGCAAGCATGTCAA
               GTAGTGACCCGTCACGTAAATTTGTCGGATGAAGACCTT
               AAAACTAAAGTAATTAATATCGTGAAGGAAACGAAAAA
               GATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGT
               TGGTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGC
               ACTGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGT
               CGTGGGCGGCTCGCGCGCAGTGATTGATTCCGGCTGGTT
               ACCTGCGGATCATCAGGTTGGACAAACCGGTAAGACCGT
               GCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGGGC
               TATCCAGCATAAGGCTGGGATGCAAGACTCTGAACTGAT
               CATTGCCGTCAACAAAGACGAAACGGCGCCTATCTTCGA
               CTGCGCCGATTATGGCATCACCGGTGATTTATTTAAAATC
               GTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAA
               CGCATGAtaagaaggagatatacatATGCGCATCTATGTGTGTGTG
               AAACAAGTCCCAGATACGAGCGGCAAGGTGGCCGTTAA
               CCCTGATGGGACCCTTAACCGTGCCTCAATGGCAGCGAT
               TATTAACCCGGACGATATGTCCGCGATCGAACAGGCATT
               AAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC
               TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCG
               AAATTATTGCAATGGGGGCCGACGATGGTGTGCTGATTT
               CGGCCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCA
               GTCAAATTATTAGCGCGGCAATCCATAAATTAGGCTTAA
               GCAATGAAGACATGATCTTTTGCGGTCGTCAGGCCATTG
               ACGGTGATACGGCCCAAGTCGGCCCTCAAATTGCCGAAA
               AACTGAGCATCCCACAGGTAACCTATGGCGCAGGAATCA
               AAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGTTGG
               AGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTC
               TGATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTT
               ACATGACTCTCAACGGTATTATGGAATGCTACTCCAAGC
               CGCTCCTCGTTCTCGATTACGAAGCACTGAAAGATGAAC
               CGCTGATCGAACTTGATACCATTGGGCTTAAAGGCTCCC
               CGACGAATATCTTTAAATCGTTTACGCCGCCTCAGAAAG
               GCGTTGGTGTCATGCTCCAAGGCACCGATAAGGAAAAAG
               TCGAGGATCTGGTGGATAAGCTGATGCAGAAACATGTCA
               TCTAAtaagaaggagatatacatATGTTCTTACTGAAGATTAAAAA
               AGAACGTATGAAACGCATGGACTTTAGTTTAACGCGTGA
               ACAGGAGATGTTAAAAAAACTGGCGCGTCAGTTTGCTGA
               GATCGAGCTGGAACCGGTGGCCGAAGAGATTGATCGTG
               AGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGCGG
               AAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTG
               GTGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCG
               TGTCAGAATTCGGCAAAAAGTGTATGGCCTCAGCTTCCA
               TTTTAAGCATTCATCTTATCGCGCCGCAGGCAATCTACAA
               ATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGC
               GTCTTACCAAAGGTGGTGAACTGGGCGCCTTTGCGCTGA
               CAGAACCAAACGCCGGAAGCGATGCCGGCGCGGTAAAA
               ACGACCGCGATTCTGGACAGCCAGACAAACGAGTACGT
               GCTGAATGGCACCAAATGCTTTATCAGCGGGGGCGGGCG
               CGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAA
               AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAA
               AGGGACCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCA
               AGATGGGGATCGCAGGTTCGGAAACCGCGGAACTTATCT
               TCGAAGATTGTCGCGTTCCGGCTGCCAACCTTTTAGGTA
               AAGAAGGCAAAGGCTTTAAAATTGCTATGGAAGCCCTGG
               ATGGCGCCCGTATTGGCGTGGGCGCTCAAGCAATCGGAA
               TTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGTTC
               ACGAGCGCATTCAATTTGGTAAACCGATCGCGAATCTGC
               AGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAA
               CCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATC
               TTGAAGACGCGGGTAAACCGTTCACAAAGGAATCTGCTA
               TGTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGA
               CAAATTTAGCTCTGCAGATTCACGGGGGTTACGGTTATA
               TGAAAGATTATCCGTTAGAGCGTATGTATCGCGATGCTA
               AGATTACGGAAATTTACGAGGGGACATCAGAAATCCATA
               AGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA
Ptet-acuI-pct- caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggg
lcdABC         gatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaac
(Ptet: lower   gacggccagtgaattgacgcgtattgggatgtaaaacgacggccagtgaattcgttaagaccc
case; tetA/R   actttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggc
promoter       tctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagta
within Ptet:   ggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatg
lower case     ccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgaggcataaaaaggcta
bold, with tet attgattttcgagagtttcatactgatttctgtaggccgtgtacctaaatgtacttttgctccatcgc
operator       gatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttcccc
underlined;    ttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaa
RBS and        gcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttac
leader region  gggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttat
lower case     ctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagttattttaccac
italic;        tccctatcagtgatagaga              aaagtgaactctagaaataattttgtttaactttaagaaggaga
ribosome       tatacat              ATGCGTGCGGTACTGATCGAGAAGTCCGATGATAC
binding site:  ACAGTCCGTCTCTGTCACCGAACTGGCTGAAGATCAACT
lower case     GCCGGAAGGCGACGTTTTGGTAGATGTTGCTTATTCAAC
underlined     ACTGAACTACAAAGACGCCCTGGCAATTACCGGTAAAGC
italic; coding CCCCGTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGAC
region: upper  TTTACGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCA
case, rrnB T1  AGCCAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGG
and T2         GGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGC
terminors:     GTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTG
lower case     GACTTACGCCAAGCGGCCATGATCGGTACAGCAGGATAC
bold underline ACGGCGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGA
italics) (SEQ  GTGGTGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCA
ID NO: 241)    GCAGGCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCC
               GCTAAGGGCTATGAGGTAGCGGCAGTGACTGGACGTGC
               GTCCGAAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAG
               CGTAATTGATCGTAACGAATTAACGGGGAAGGTACGCCC
               GCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGC
               GGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAA
               GTATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGG
               CATGGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGT
               GGGATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCA
               AAGACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCA
               GATCTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAG
               TTGCCGTTTAGTGAAGTAATCGAGACAGCACCCAAATTC
               TTGGACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTA
               ACGCCCTAAgaactctagaaataattttgtttaactttaagaaggagatatacatAT
               GCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAA
               AACTGATCAAGGACGGCGACACCGTGACAACTAGCGGC
               TTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCA
               GTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGAA
               CATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGA
               CGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGCT
               GAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGC
               GTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCT
               ACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGA
               TATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGG
               AATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGAA
               GGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGT
               GGAAATTAAAGGGCAGGAATACCTGTTTTATCCGGCGTT
               CCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGA
               CGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCC
               TTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTC
               GGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAA
               AGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGG
               TATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGA
               CCATCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTT
               AGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACC
               ACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGG
               CGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGG
               GGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGA
               AGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAGTC
               CGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTT
               TGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGG
               CTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTT
               TGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAAT
               ATCAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCG
               GCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCT
               TCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGA
               AAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGA
               AACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCT
               TTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTA
               CCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAG
               ACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATC
               TGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGAT
               CATTGATCGTGACGCAAACGGGCAGATTAAACTGATGGA
               CGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGA
               AATGAAGTCTTGAtaagaaggagatatacatATGAGCTTAACCCA
               AGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAGGG
               TAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCG
               GTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG
               AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGG
               AGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCG
               ATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAA
               CGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTAC
               ATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAG
               CCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGC
               TTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACAC
               GCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGA
               TATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACC
               ATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGAC
               CAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTG
               GCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAG
               ATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTG
               CCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCT
               TCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCG
               CAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGC
               GGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACG
               CCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAG
               GTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAAT
               CTATGAAGAATCTGAATTCGATTATGACCGGTACGGCAT
               ACCCCGCCCTTTGGGACCTGCACTATGACGCTAACGACG
               AATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTT
               ATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGC
               TTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTAT
               ATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGA
               ACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGT
               CTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTC
               GCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGC
               CCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGG
               AATAAtaagaaggagatatacatATGTCACGCGTGGAGGCAATCC
               TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA
               GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC
               GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT
               ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC
               CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC
               TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT
               ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT
               TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG
               AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT
               CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG
               GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA
               GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT
               TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT
               GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT
               GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG
               TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA
               CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG
               GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA
               CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC
               GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGG
               ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC
               TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC
               TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG
               TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC
               GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG
               GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG
               GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG
               TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG
               AAATGCTTTAAtaagaaggagatatacatATGTATACCTTGGGGAT
               TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA
               AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT
               CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA
               AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG
               CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG
               CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC
               CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT
               ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG
               GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT
               AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG
               GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA
               CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC
               ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA
               TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT
               GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG
               TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT
               GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC
               GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC
               TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA
               TGAGGCCGCCCAGAAGAAGTAgatggtagtgtggggtctccccatgcga
               gagtagggaactgccaggcat
               ccgccgggagcggattt
               gaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgcca
               ggcatcaaattaagc
acuI-pct-      ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAG
lcdABC (SEQ    TCCGTCTCTGTCACCGAACTGGCTGAAGATCAACTGCCG
ID NO: 242)    GAAGGCGACGTTTTGGTAGATGTTGCTTATTCAACACTG
               AACTACAAAGACGCCCTGGCAATTACCGGTAAAGCCCCC
               GTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGACTTTA
               CGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCAAGC
               CAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGGGGG
               AAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGCGTGC
               GCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACTT
               ACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGG
               CGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGG
               TGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCAGCAG
               GCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCCGCTA
               AGGGCTATGAGGTAGCGGCAGTGACTGGACGTGCGTCCG
               AAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAGCGTA
               ATTGATCGTAACGAATTAACGGGGAAGGTACGCCCGCTG
               GGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCGGG
               ATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTA
               TCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCAT
               GGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGG
               ATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCAAAG
               ACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCAGAT
               CTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAGTTG
               CCGTTTAGTGAAGTAATCGAGACAGCACCCAAATTCTTG
               GACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTAACG
               CCCTAAgaactctagaaataattttgtttaactttaagaaggagatatacatATGCG
               CAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACT
               GATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGT
               GGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGA
               AAAGCGTTTCCTGGAAACGGGCGAACCGAAGAACATTA
               CTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTC
               GTGGCGCAGAACATTTCGCGCATGAAGGCCTGCTGAAAC
               GTTATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAG
               GGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAAT
               GTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATATTG
               CGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCG
               GCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCA
               ACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAA
               ATTAAAGGGCAGGAATACCTGTTTTATCCGGCGTTCCCG
               ATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAG
               AGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTG
               GAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTCGGGT
               GGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCA
               GGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTATC
               TATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCAT
               CAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGT
               GGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACT
               GCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGC
               GATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGT
               AGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGA
               AGGCATTGTGGATTTTATGACTCTCACAGCGGAGTCCGG
               CGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGG
               GGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTA
               CCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGT
               TACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATC
               AATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGC
               GGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTC
               TTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGAAA
               ATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAA
               CAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTT
               AATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTAC
               CTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGA
               CGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCT
               GCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGAT
               CATTGATCGTGACGCAAACGGGCAGATTAAACTGATGGA
               CGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGA
               AATGAAGTCTTGAtaagaaggagatatacatATGAGCTTAACCCA
               AGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAGGG
               TAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCG
               GTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG
               AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGG
               AGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCG
               ATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAA
               CGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTAC
               ATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAG
               CCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGC
               TTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACAC
               GCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGA
               TATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACC
               ATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGAC
               CAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTG
               GCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAG
               ATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTG
               CCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCT
               TCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCG
               CAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGC
               GGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACG
               CCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAG
               GTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAAT
               CTATGAAGAATCTGAATTCGATTATGACCGGTACGGCAT
               ACCCCGCCCTTTGGGACCTGCACTATGACGCTAACGACG
               AATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTT
               ATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGC
               TTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTAT
               ATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGA
               ACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGT
               CTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTC
               GCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGC
               CCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGG
               AATAAtaagaaggagatatacatATGTCACGCGTGGAGGCAATCC
               TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA
               GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC
               GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT
               ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC
               CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC
               TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT
               ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT
               TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG
               AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT
               CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG
               GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA
               GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT
               TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT
               GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT
               GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG
               TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA
               CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG
               GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA
               CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC
               GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGG
               ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC
               TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC
               TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG
               TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC
               GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG
               GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG
               GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG
               TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG
               AAATGCTTTAAtaagaaggagatatacatATGTATACCTTGGGGAT
               TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA
               AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT
               CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA
               AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG
               CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG
               CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC
               CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT
               ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG
               GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT
               AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG
               GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA
               CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC
               ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA
               TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT
               GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG
               TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT
               GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC
               GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC
               TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA
               TGAGGCCGCCCAGAAGAAGTA

Example 26

Quantification of Propionate by LC-MS/MS

Sample Preparation

First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 m/mL sodium propionate standards were prepared in water. Then, 25 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 75 μL of 60% ACN (45 uL ACN+30 uL water per reaction) with 10 ug/mL of propionate-d5 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 5 μL of diluted samples were added to 95 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour

LC-MS/MS Method

Propionate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 49 and Table 50. Tandem Mass Spectrometry details are found in Table 51.

TABLE 49
HPLC Details
Column    Thermo Aquasil C18
          column, 5 μm (50 × 2.1 mm)
Mobile    100% H2O, 0.1% Formic
Phase A   Acid
Mobile    100% ACN, 0.1% Formic
Phase B   Acid
Injection 10 uL
volume

TABLE 50
HPLC Method
	Total	Flow
	Time	Rate
	(min)	(μL/min)	A %	B %
	0	0.5	100	0
	1	0.5	100	0
	2	0.5	10	90
	4	0.5	10	90
	4.01	0.5	100	0
	4.25	0.5	100	0

TABLE 51
Tandem Mass Spectrometry Details
Ion Source  HESI-II
Polarity    Positive
SRM         Propionate
transitions 156.2/57.1,
            Propionate-d5
            161/62.1

Example 27

GLP-1 Production from Genetically Engineered Bacteria and Activity Measurements

To determine whether GLP-1 can be expressed by the genetically engineered bacteria, a construct expressing GLP-1 in conjunction with a modified flagellar type III secretion system shown in FIG. 61 was generated and integrated into the E coli Nissle chromosome. The construct comprises GLP-1 under control of the native FliC promoter and 5′UTR (untranslated region containing the N-terminal flagellar secretion signal) with an optimized ribosome binding site FIG. 23 and Table 52).

TABLE 52
GLP-1 construct sequences
Description and SEQ ID NO        Sequence
GLP-1 under control of the native ttaaccacgacctttaaccagccaagcaataaactctttcgcagc
FliC promoter and 5′UTR with an  ctggccctccaaatagctagaaacatcagaagtgaaagttccct
optimized ribosome binding site (in ccgcgtggcgttcgaactcgtccatattacctcctgactgtgtcta
reverse orientation)             cttcgttgattacgttttgggtttccacccgtcggctcaatcgccgt
(SEQ ID NO: 243)                 ca
GLP-1 (in reverse orientation)   ttaaccacgacctttaaccagccaagcaataaactctttcgcagc
(SEQ ID NO: 244)                 ctggccctccaaatagctagaaacatcagaagtgaaagttccct
                                 ccgcgtggcgttcgaactcgtccat
FliC 5′ UTR (in reverse orientation) attacctcctgactgtgtctacttcgttgattacgttttgggtttcca
(SEQ ID NO: 245)                 cccgtcggctcaatcgccgtca
Optimized RBS (in reverse        attacctcctgactgtgtctacttc
orientation)
(SEQ ID NO: 246)
Putative terminator              gggcagaaaaaaccccgccgttggcggggaagcacgttgc
(SEQ ID NO: 247)
GLP-1 construct comprising       Gggcagaaaaaaccccgccgttggcggggaagcacgttgc
terminator (lower case italic) GLP-1 tggcaaattaccattcatgttgccggatgcggcgtaaacgcctta
(lower case bold) under control of tccggcctacaaaaatgtgcaaattcaataaattgcaattcccctt
the native FliC promoter and 5′UTR gtaggcctgataagcgcagcgcatcaggcaatttggcgttgcc
(upper case bold, with optimized gtcagtctcagttaatcaggttacggcgattaaccacgaccttt
RBS underlined) and a            aaccagccaagcaataaactctttcgcagcctggccctcca
chloramphenicol resistance gene  aatagctagaaacatcagaagtgaaagttccctccgcgtgg
under the control of the cat promoter cgttcgaactcgtccat              ATTACCTCCTGACTGTG
(upper case italic bold), frt homology TCTACTTCGTTGATTACGTTTTGGGTTT
(upper case underlined)          CCACCCGTCGGCTCAATCGCCGTCAAC
(SEQ ID NO: 248)                 CCTGTTATCGTCTGTCGTAAAACAACC
                                 TTTAGAATTTTTTTCACAAACAGCCATT
                                 TTTTGTTAGTCGACGAAATACTCTTTTC
                                 TCTGCCCCTTATTCCCGCTATTAAAAAA
                                 AACAATTAAACGTAAACTTTGCGCAAT
                                 TCAGGCCGATAACCCCGGTATTCGTTT
                                 TACGTGTCGAAAGATAAACGAAGTTCC
                                 TATACTTTCTAGAGAATAGGAACTTCG
                                 GAATAGGAACTTCATTTCTCGTTCGCT
                                 GCCACCTAAGAATACTCTACGGTCACA
                                 TACAAATGGCGCGCCTTACGCCCCGCC
                                 CTGCCA
                                 ACG
                                 TCTCATTTTCGCCAAAAGTTGGCCCAG
                                 GGCTTCCCGGTATCAACAGGGACACCA
                                 GGATTTATTTATTCTGCGAAGTGATCTT
                                 CCGTCACAGGTAGGCGCGCCGAAGTTC
                                 CTATACTTTCTAGAGAATAGGAACTTC
                                 GGAATAGGAACT

Cultures (the genetically engineered bacteria comprising the GLP-1 construct or streptomycin resistant control Nissle) are grown overnight in F-12K medium (Mediatech, Manassas, Va.) without glucose (containing selective antibiotics (chloramphenicol or streptomycin) and then diluted 1:200. The cells are grown with shaking at 250 rpm, and at indicated times (0, 3, 6, and 12 h), the supernatant aliquots are collected for GLP-1 quantification.

Additionally, bacteria are pelleted, washed, and harvested, resuspended in 25 mL sonication buffer (50 mM Tris-HCl, 30 mM NaCl, pH 8.0) with protease inhibitors, and lysed by sonication on ice. Insoluble debris is spun down twice for 20 min at 12,000 rpm at 4° C. to detect any intracellular recombinant protein.

To generate cell free medium, the supernatant is centrifuged, and filtered through a 0.2-micron filter to remove any remaining bacteria. The cell-free culture medium (CFM) is diluted to OD600=1 with F-12K, and 10 ng/ml leupeptin, 200 μM PMSF and 5 ng/mL aprotinin was added to the CFM to inhibit proteases prior storage at 4° C.

Western Blotting

The cell-free culture medium (CFM) was diluted to the same OD600 with F-12K, and 10 ng/ml leupeptin, PMSF and 5 ng/mL aprotinin was added to inhibit proteases. Clarified supernatant (14 ml) is precipitated with 10% trichloroacetic acid (TCA, VWR) for 30 min on ice, and the pellet was washed twice in ice-cold ethanol/ether (1:1). The supernatant pellet is dried under vacuum, dissolved in 50 pi sample buffer (2% SDS, 50 mM Tris, pH 6.8, 20%glycerol, 10% mercaptoethanol, bromophenol blue) and boiled for 5 min at 95° C. The cell pellet is resuspended (From 14 ml culture) in room temperature BugBuster Master Mix by gentle vortexing, using 500 μl BugBuster Master Mix with protease inhibitors (10 ng/ml Leupeptin, 200₁IMPMSF and 5 ng/mL aprotinin). The cell suspension is incubated on a shaking platform (VWR, Bristol, Conn.) at a slow setting for 10-20 min at room temperature. 125 μl 5× sample buffer is added to each sample before and boiling for 10 min at 95° C.

Protein concentration is determined by BCA protein assay, and isolated proteins are analyzed by Western blot. Proteins are transferred onto PVDF membranes are detected with an HRP-conjugated Glucagon Antibody (24HCLC), ABfinity™ Rabbit Oligoclonal, Thermo Fisher.

Co-culture with Caco-2 cells and ELISA for Insulin

To determine whether the GLP-1 expressed by the genetically engineered bacteria is functional, a co-culture experiment is conducted in which the bacterial supernatant containing GLP-1 is added to the growth medium of a mammalian intestinal cell line, Caco-2. Caco-2 cells are an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions, and which lends itself to the in vitro study of human gut. The ability of the Caco-2 cells to produce insulin upon exposure to the bacterial cell free supernatant is measured.

Caco-2 epithelial cells (ATCC# CRL-2102, Manassas, Va.) are maintained in Dulbecco's Modified Eagle Media (DMEM, Cellgro, Herndon,Va.) plus 10% FBS (Cellgro) at 37° C. in a humidified incubator supplemented with 5% CO2. For co-culture experiments, Caco-2 cells are grown in F-12K supplemented with 10% FBS at 37° C. in a humidified incubator supplemented with 5% CO2. All co-culture experiments are performed in F-12K plus 10% FBS with Caco-2 cells in passages between 15 and 22.

Approximately 80% confluent monolayers of Caco-2 cells in 12-well plates are washed with fresh F-12K plus 10% FBS once and covered with 1 mL 50% CFM in F-12K with 10% FBS and incubated at 37° C. with 5% CO₂. 200 nM. As a control, the same volume of recombinant GLP-1 (200 nM) in F-12K with 10% FBS is added as a positive control in separate wells. Following a 16 h incubation, an additional 1 mL of 50% CFM in F-12K with 10% FBS or GLP-1 is added to the cells, supplemented with 0.4% Glucose or 0.4% Glycerol before incubation for an additional 2 h. The media is removed from the cells, supplemented with Leupeptin (10 ng/mL), 0.2 mM PMSF and aprotinin (10 ng/mL), centrifuged (12,000×rpm), and kept briefly at 4° C. prior to ELISA analysis for insulin expression (see “Immuno-blot and ELISA” section).

In order to estimate the amount of insulin secreted from Caco-2 cells activated by Glp-1, cell free supernatants are assayed using standard ELISA procedures using the Insulin ELISA Kit, Human (KAQ125, Thermo Fisher), according to manufacturer's instructions.

Example 28

In Vivo NASH Studies

For in vivo studies, a mouse model is used to study the effects of liver steatosis and hepatic inflammation (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice are fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed. A decrease in liver damage after treatment with the engineered bacterial cells indicates that the engineered bacterial cells described herein are effective for treating nonalcoholic steatohepatitis (NASH).

Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with nonalcoholic steatohepatitis (NASH), for example, weight loss, further indicates the efficacy of the engineered bacterial cells described herein for treating nonalcoholic steatohepatitis (NASH).

Example 29

Construction of Plasmids Encoding Bile Salt Hydrolase Enzymes

The bile salt hydrolase genes from Lactobacillus plantarum (SEQ ID NO:1) is synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57-Kan by Gibson assembly, and transformed into E. coli DH5α as described herein to generate the plasmid pTet-BSH.

Example 30

Generation of Recombinant Bacteria Comprising a Bile Salt Hydrolase Enzyme

The pTet-BSH plasmid described above is transformed into E. coli Nissle, DH5α, or PIR1. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli (Nissle, DH5α or PIR1) is diluted 1:100 in 4 mL of LB and grown until it reaches an OD₆₀₀ of 0.4-0.6. 1mL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10% glycerol. The electroporator is set to 1.8 kV. 1 uL of a pTet-BSH miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500 uL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing 50 ug/mL Kanamycin for pTet-BSH.

Example 31

Functional Assay Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration

For in vitro studies, all incubations will be performed at 37° C. Cultures of E. coli Nissle containing pTet-BSH are grown overnight in LB and then diluted 1:100 in LB. The cells are grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of bile salt hydrolase, and bacteria are grown for another 3 hours. Culture broths are then inoculated at 20% in flasks containing fresh LB culture media containing excess bile salts (either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile) and grown for 16 hours with shaking (250 rpm). A “medium blank” for each culture condition broth is also prepared whereby the “medium blank” is not inoculated with bacteria but treated under the same conditions as the inoculated broths. Following the 16 hour incubation period, broth cultures are pasteurized at 90° C. for 15 minutes, centrifuged at 5,000 rpm for 10 minutes, and supernatants filtered with a 0.45 micron filter.

Bile salt levels and activity in the supernatants is determined. Briefly, bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner, Applied Environ. Microbiol., 55:11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992). BSH activity can also be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al., PNAS, 105(36):13580-13585 (2008)). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3):186-202 (2012)).

Example 32

In Vivo Studies Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration

For in vivo studies, a mouse model of weight gain and lipid metabolism (as described by Joyce et al., PNAS, 111(20):7421-7426 (2014)) is used. To briefly summarize, C57BL/6J mice and germ-free Swiss Webster mice can be fasted and fed either a normal low-fat diet or a high-fat diet for ten weeks. After ten weeks, the mice can be inoculated with recombinant bacteria comprising a bile salt hydrolase enzyme (as described herein) or control bacteria. Body weight, plasma samples, and fecal samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and internal organs (liver, spleen, intestines) and fat pads can be removed and assayed. Treatment efficacy is determined, for example, by measuring levels of bile salts and bile acids. A decrease in levels of bile salts after treatment with the recombinant bacterial cells indicates that the recombinant bacterial cells described herein are effective for treating disorders associated with bile salts.

Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with disorders associated with bile salts, for example, weight loss, further indicates the efficacy of the recombinant bacterial cells described herein for treating disorders associated with bile salts.

Example 33

Nitric oxide-Inducible Reporter Constructs

ATC and nitric oxide-inducible reporter constructs were synthesized (Genewiz, Cambridge, Mass.). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (FIG. 77); promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are

TABLE 53
SEQ ID NO: 249
ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgt
tgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccg
ccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccat
acactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgtt
cgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccg
cctttgaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcga
ggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgc
aattttaaactctagaaataattttgtttaactttaagaaggagatatacata                              tggcta
gcaaaggcgaagaattgttcacgggcgttgttcctattttggttgaattggatggcgat
gttaatggccataaattcagcgttagcggcgaaggcgaaggcgatgctacgtatggcaa
attgacgttgaaattcatttgtacgacgggcaaattgcctgttccttggcctacgttgg
ttacgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaaacgt
catgatttcttcaaaagcgctatgcctgaaggctatgttcaagaacgtacgattagctt
caaagatgatggcaattataaaacgcgtgctgaagttaaattcgaaggcgatacgttgg
ttaatcgtattgaattgaaaggcattgatttcaaagaagatggcaatattttgggccat
aaattggaatataattataatagccataatgtttatattacggctgataaacaaaaaaa
tggcattaaagctaatttcaaaattcgtcataatattgaagatggcagcgttcaattgg
ctgatcattatcaacaaaatacgcctattggcgatggccctgttttgttgcctgataat
cattatttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcatat
ggttttgttggaattcgttacggctgctggcattacgcatggcatggatgaattgtata
aa              taataa

These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600=˜0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

FIG. 77 shows NO-GFP constructs (the dot blot) E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1:100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using

Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS-treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1:100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. FIG. 77D shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Example 34

FNR Promoter Activity

In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 18 and Table 19. The nucleotide sequences of these constructs are shown in Tables 54-58 ((SEQ ID NO: 250-254). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in FIG. 75.

Table 54 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_fnr1 (SEQ ID NO: 250). The construct comprises a translational fusion of the Nissle nirB1 gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The P_fnri sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 55 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_fnr2 ((SEQ ID NO: 251). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The P_fnr2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 56 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_fnr3 ((SEQ ID NO: 252). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The P_fnr3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 57 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_fnr4 ((SEQ ID NO: 253). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The P_fnr4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 58 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS ((SEQ ID NO: 254). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrS1, fused to lacZ. The P_fnrs sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

TABLE 54
Pfnr1-lacZ construct Sequences
Nucleotide sequences of Pfnr1-lacZ construct, low-copy
(SEQ ID NO: 250)
GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcacta
tcgtcgtccggccttttcctctcttactctgctacgtacatctatttctataaatcc
gttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgacttaa
gaaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaatttga
tttacatcaataagcggggttgctgaatcgttaaggtaggcggtaatag                              aaaagaaa
tcgaggcaaaa                            ATGagcaaagtcagactcgcaattatGGATCCTCTGGCCGTCGTAT
TACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCAC
ATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC
AACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAG
CGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCC
CCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATC
CCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGC
TCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG
ATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCC
AGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAA
ACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGG
ATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCA
CGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGG
TACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGG
TTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTG
AAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTG
AAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAAC
TGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCG
AGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCG
GCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGA
TGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGC
ATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGG
TGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATG
ATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATC
GTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTA
ATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGT
ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGC
GCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGC
TTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGG
GTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTT
TACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAA
ACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCC
AGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGG
AAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAG
TGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGG
CACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAG
GTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCT
GGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACA
TCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCG
CGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGG
GTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTG
GCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGG
ATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAAC
GCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAG
ATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGA
AAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCA
TCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA
CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAG
AAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGT
CAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGC
GCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCC
GCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAG
AAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCT
GGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGT
TGGTCTGGTGTCAAAAATAA

TABLE 55
Pfnr2-lacZ construct sequences
Nucleotide sequences of Pfnr2-lacZ construct, low-copy
(SEQ ID NO: 251)
GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctc
atgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacag
gagtatttatattgcgcccgttacgtgggcttcgactgtaaatc                              agaaaggagaaaaca
cct                            ATGacgacctacgatcgGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGA
AAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGC
GTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC
GAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTG
CGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACG
ATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCC
GCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACA
GGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCA
ACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGC
GCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGG
CAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGT
TGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGAT
GATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGA
ACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGC
CTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGC
CTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGT
GGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTT
TCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATT
CGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGAC
GATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGC
ATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTG
GATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCC
GCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATC
ACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGAC
GCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGG
CGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAG
ACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGA
GAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGG
CTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCT
GGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCT
TACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGT
CTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATT
TCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCAT
AGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGG
TGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGC
CGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCG
ACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAA
CCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAA
CGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTT
CTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTT
CACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTA
ACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTG
CAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCA
GCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTG
AGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATT
GGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC
GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCAT
TGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACG
CGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCG
CTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAG
GCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGC
CCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTG
GTGTCAAAAATAA

TABLE 56
Pfnr3-lacZ construct Sequences
Nucleotide sequences of Pfnr3-lacZ construct, low-copy
(SEQ ID NO: 252)
GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcact
atcgtcgtccggccttttcctctcttactctgctacgtacatctatttctataaat
ccgttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgact
taagaaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaat
ttgatttacatcaataagcggggttgctgaatcgttaaGGATCCctctagaaataa
ttttgtttaactttaagaaggagatatacat                            ATG              ACTATGATTACGGATTCTCTGG
CCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC
CTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA
TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTC
CGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGAT
ACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACAC
CAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGA
CAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAG
ACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCG
CTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCAT
TTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGC
AGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTC
GTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTA
ATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTG
CGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAG
CGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATC
GCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCG
AATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGC
AGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGC
TGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTG
CATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCA
GAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACA
CGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCAC
GGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAG
CGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCT
GGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGG
ATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACAC
CACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCT
TCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATG
CGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTT
CGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCT
GGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCG
GCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGG
TCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAAC
AGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATAC
CTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAA
GCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGA
TTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTA
CGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTG
GCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACG
CCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAG
CGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGA
AAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACG
ACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGG
AAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATAC
ACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA
CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATC
AATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGAC
CTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAG
AAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTG
TCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGAC
GCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCA
GCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCG
GAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGA
CTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATT
ACCAGTTGGTCTGGTGTCAAAAATAA

TABLE 57
Pfnr4-lacZ construct Sequences
Nucleotide sequences of Pfnr4-lacZ construct, low-copy
(SEQ ID NO: 253)
GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatgg
ctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcact
cgacaggagtatttatattgcgcccGGATCCctctagaaataattttgtttaactt
taagaaggagatatacat                            ATG              ACTATGATTACGGATTCTCTGGCCGTCGTATTACA
ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATC
CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA
CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGC
GGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCC
CCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTAT
CCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTC
GCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTT
TTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTAC
GGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGG
AGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAG
ATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAA
CCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAG
CCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGC
GGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCT
TTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACG
CCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTG
CAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGAC
GTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCC
GTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCA
TGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAAC
GCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCG
CTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAA
TGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACG
CGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAA
TGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCG
ATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGAT
ATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCC
GAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCC
TTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGG
CAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGA
TCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTG
ATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCC
GACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCA
GTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATA
GCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGC
GGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGA
ACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAAC
CAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGT
CTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACT
GACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTA
ACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTG
ACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAG
TGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCC
ATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCG
GTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAG
CCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTG
CGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCG
CAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGA
CCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATA
CCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAAT
TATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCA
ACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACAT
GGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCG
TCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTG
GTGTCAAAAATAA

TABLE 58
Pfnrs-lacZ construct Sequences
Nucleotide sequences of Pfnrs-lacZ construct, low-copy
(SEQ ID NO: 254)
GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgt
aacaaaagcaatttttccggctgtctgtatacaaaaacgccgtaaagtttgagcgaagt
caataaactctctacccattcagggcaatatctctcttGGATCCctctagaaataattt
tgtttaactttaagaaggagatatacat                            ATG              CTATGATTACGGATTCTCTGGCCGTCGT
ATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCAC
ATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA
CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGT
GCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAA
ACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACG
GTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAA
TATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACT
CGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTG
CCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGAT
GGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCG
GCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA
GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGAT
GTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGC
AGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTAT
GCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAAT
CCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAG
CAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTG
AACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGG
TCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACT
TTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGAC
CGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAAT
GAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGA
TGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCA
GGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCG
CCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGA
TGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAA
AAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGC
GATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCC
GTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAA
AACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCA
GTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAG
CAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACC
AGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGA
TGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGT
TGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTA
CGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCA
GCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCC
CTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAA
TTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCT
GACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTG
AAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTAC
CAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGAT
TACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCT
ACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGAT
ACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGT
AAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTT
TTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAA
AACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGA
CTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCC
ATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATT
GGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCG
CTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Example 35

Increasing in vitro Butyrate and Acetate Production in Engineered Nissle

E. coli generates high levels of acetate as an end product of fermentation. In order to improve acetate production while also maintaining high levels butyrate production, deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and 1 dh (lactate dehydrogenase) were generated to prevent or reduce metabolic flux through pathways which do not result in acetate or butyrate production (see, e.g., FIG. 14). For this study, Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes were used. Additionally, for this study media M9 media containing 50 mM MOPS with 0.5% glucose was comparedto media containing 0.5/% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut.

Briefly, bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein in Example 18 and Example 21.

As seen in FIG. 14A and FIG. 14B, both integrated strains made similar amounts of acetate, and FNRS-ter-pbt-buk butyrate cassettes produced slightly more butyrate. Deletions in adhE and ldhA have similar effects on butyrate and acetate production. Acetate production was much greater in media containing 0.5% glucuronic acid.

In alternate embodiments, frd (fumarate reductase) is deleted to assess the effect of the deletion on acetate and butyrate production.

Example 36

Generation of Indole Propionic Acid Strain and In Vitro indole Production

To facilitate inducible production of indole propionic acid (IPA) in Escherichia coli Nissle, 6 genes allowing the production of indole propionic acid from tryptophan, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 under a tet inducible promoter. In other embodiments, the IPA synthesis cassette is put under the control of an FNR, RNS or ROS promoter, e.g., described herein, or other promoter induced by conditions in the healthy or diseased gut, e.g., inflammatory conditions. For efficient translation of IPA synthesis genes, each synthetic gene in the cassette is separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

The IPA synthesis cassette comprises TrpDH (tryptophan dehydrogenase from Nostoc punctiforme NIES-2108), FldH1/F1dH2 (indole-3-lactate dehydrogenase from Clostridium sporogenes), FldA (indole-3-propionyl-CoA: indole-3-lactate CoA transferase from Clostridium sporogenes), FldBC (indole-3-lactate dehydratase from Clostridium sporogenes), FldD (indole-3-acrylyl-CoA reductase from Clostridium sporogenes), and Acul (acrylyl-CoA reductase from Rhodobacter sphaeroides).

The tet inducible IPA construct described above is transformed into E. coli Nissle as described herein and production of IPA is assessed. In certain embodiments, E. coli Nissle strains containing the IPA synthesis cassette described further comprise a tryptophan synthesis cassette. In certain embodiments, the strains comprise a feedback resistant version of AroG and TrpE to achieve greater Trp production. In certain embodiments, additionally, the tnaA gene (tryptophanase converting Trp into indole) is deleted.

All incubations are performed at 37° C. LB-grown overnight cultures of E. coli Nissle transformed with the IPA biosynthesis construct alone or in combination with a tryptophan biosynthesis construct and feedback resistant AroG and TrpE are subcultured 1:100 into 10 mL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of the IPA biosynthesis and tryptophan biosynthesis constructs. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (e.g., 0 up to 24 hours) by LC-MS to assess levels of IPA.

Production of IPA is also assessed in E. coli Nissle strains containing the IPA and tryptophan cassettes both driven by an RNS promoter e.g., a nsrR-norB-IPA biosynthesis construct) in order to assess nitrogen dependent induction of IPA production. Overnight bacterial cultures are diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (0 up to 24 hours) to assess IPA levels.

In alternate embodiments, production of IPA is also assessed in E. coli Nissle strains containing the IPA and tryptophan cassettes both driven by the low oxygen inducible FNR promoter, e.g., FNRS, or the reactive oxygen regulated OxyS promoter.

Example 37

Synthesis of Constructs for Synthesis of Tryptophan, Tryptamine, and Other Indole Metabolites

Various constructs were synthesized, and cloned into vector pBR322 for transformation of E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome according to methods described herein, e.g., Example 2.

TABLE 59
Sequences
Description        Sequence
fbrAroG (RBS and   Ctctagaaataattttgtttaactttaagaaggagatatacat
leader region      atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg
underlined)        cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga
SEQ ID NO: 255     aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca
                   tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg
                   aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta
                   cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac
                   gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg
                   gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc
                   gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc
                   ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta
                   atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt
                   gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa
                   ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc
                   agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat
                   ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt
                   gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct
                   ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt
                   acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa
fbrAroG            atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg
SEQ ID NO: 256     cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga
                   aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca
                   tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg
                   aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta
                   cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac
                   gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg
                   gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc
                   gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc
                   ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta
                   atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt
                   gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa
                   ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc
                   agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat
                   ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt
                   gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct
                   ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt
                   acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa
fbrAroG-serA       Ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacg
(RBS and leader    atttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattcccc
region underlined; gctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct
SerA starts after  gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtc
second RBS)        gcggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagct
SEQ ID NO: 257     ggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggc
                   tgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc
                   gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggata
                   tgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtacca
                   ccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa
                   atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgc
                   actgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg
                   gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt
                   gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat
                   ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg
                   ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg
                   gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc
                   accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca
                   gtaaaagcgcgtcgcgggtaaTACT
                   taagaaggagatatacatatggcaaaggtatcgctggagaaagacaagattaagtttctg
                   ctggtagaaggcgtgcaccaaaaggcgctggaaagccttcgtgcagctggttacacca
                   acatcgaatttcacaaaggcgcgctggatgatgaacaattaaaagaatccatccgcgat
                   gcccacttcatcggcctgcgatcccgtacccatctgactgaagacgtgatcaacgccgc
                   agaaaaactggtcgctattggctgtttctgtatcggaacaaatcaggttgatctggatgcg
                   gcggcaaagcgcgggatcccggtatttaacgcaccgttctcaaatacgcgctctgttgc
                   ggagctggtgattggcgaactgctgctgctattgcgcggcgtgccagaagccaatgcta
                   aagcgcatcgtggcgtgtggaacaaactggcggcgggttcttttgaagcgcgcggcaa
                   aaagctgggtatcatcggctacggtcatattggtacgcaattgggcattctggctgaatcg
                   ctgggaatgtatgtttacttttatgatattgaaaacaaactgccgctgggcaacgccactca
                   ggtacagcatctttctgacctgctgaatatgagcgatgtggtgagtctgcatgtaccagag
                   aatccgtccaccaaaaatatgatgggcgcgaaagagatttcgctaatgaagcccggctc
                   gctgctgattaatgcttcgcgcggtactgtggtggatattccagcgctgtgtgacgcgctg
                   gcgagcaaacatctggcgggggcggcaatcgacgtattcccgacggaaccggcgac
                   caatagcgatccatttacctctccgctgtgtgaattcgacaatgtccttctgacgccacaca
                   ttggcggttcgactcaggaagcgcaggagaatatcggcttggaagttgcgggtaaattg
                   atcaagtattctgacaatggctcaacgctctctgcggtgaacttcccggaagtctcgctgc
                   cactgcacggtgggcgtcgtctgatgcacatccacgaaaaccgtccgggcgtgctaact
                   gcgctcaacaaaatttttgccgagcagggcgtcaacatcgccgcgcaatatctacaaact
                   tccgcccagatgggttatgtagttattgatattgaagccgacgaagacgttgccgaaaaa
                   gcgctgcaggcaatgaaagctattccgggtaccattcgcgcccgtctgctgtactaa
SerA               atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgca
SEQ ID NO: 258     ccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaag
                   gcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctg
                   cgatcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctat
                   tggctgtttctgtatcggaacaaatcaggttgatctggatgcggcggcaaagcgcgggat
                   cccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcga
                   actgctgctgctattgcgcggcgtgccagaagccaatgctaaagcgcatcgtggcgtgt
                   ggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcgg
                   ctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttactt
                   ttatgatattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgac
                   ctgctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaat
                   atgatgggcgcgaaagagatttcgctaatgaagcccggctcgctgctgattaatgcttcg
                   cgcggtactgtggtggatattccagcgctgtgtgacgcgctggcgagcaaacatctggc
                   gggggcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacc
                   tctccgctgtgtgaattcgacaatgtccttctgacgccacacattggcggttcgactcagg
                   aagcgcaggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaatg
                   gctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgt
                   cgtctgatgcacatccacgaaaaccgtccgggcgtgctaactgcgctcaacaaaattttt
                   gccgagcagggcgtcaacatcgccgcgcaatatctacaaacttccgcccagatgggtt
                   atgtagttattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatg
                   aaagctattccgggtaccattcgcgcccgtctgctgtactaa
fbrAroG-Tdc (tdc   ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga
from C. roseus);   tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg
RBS and leader     ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg
region underlined  aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc
SEQ ID NO: 259     ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg
                   aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg
                   attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc
                   aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg
                   atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac
                   cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat
                   ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca
                   ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg
                   gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt
                   gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat
                   ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg
                   ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg
                   gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc
                   accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca
                   gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGGGTTC
                   TATTGACTCGACGAATGTGGCCATGTCTAATTCTCCT
                   GTTGGCGAGTTTAAGCCCCTTGAAGCAGAAGAGTTCC
                   GTAAACAGGCACACCGCATGGTGGATTTTATTGCGGA
                   TTATTACAAGAACGTAGAAACATACCCGGTCCTTTCC
                   GAGGTTGAACCCGGCTATCTGCGCAAACGTATTCCCG
                   AAACCGCACCATACCTGCCGGAGCCACTTGATGATAT
                   TATGAAGGATATTCAAAAGGACATTATCCCCGGAAT
                   GACGAACTGGATGTCCCCGAACTTTTACGCCTTCTTC
                   CCGGCCACAGTTAGCTCAGCAGCTTTCTTGGGGGAAA
                   TGCTTTCAACGGCCCTTAACAGCGTAGGATTTACCTG
                   GGTCAGTTCCCCGGCAGCGACTGAATTAGAGATGATC
                   GTTATGGATTGGCTTGCGCAAATTTTGAAACTTCCAA
                   AAAGCTTTATGTTCTCCGGAACCGGGGGTGGTGTCAT
                   CCAAAACACTACGTCAGAGTCGATCTTGTGCACTATT
                   ATCGCGGCCCGTGAACGCGCCTTGGAAAAATTGGGC
                   CCTGATTCAATTGGTAAGCTTGTCTGCTATGGGTCCG
                   ATCAAACGCACACAATGTTTCCGAAAACCTGTAAGTT
                   AGCAGGAATTTATCCGAATAATATCCGCCTTATCCCT
                   ACCACGGTAGAAACCGACTTTGGCATCTCACCGCAG
                   GTACTTCGCAAGATGGTCGAAGACGACGTCGCTGCG
                   GGGTACGTTCCCTTATTTTTGTGTGCCACCTTGGGAA
                   CGACATCAACTACGGCAACAGATCCTGTAGATTCGCT
                   GTCCGAAATCGCAAACGAGTTTGGTATCTGGATTCAT
                   GTCGACGCCGCATATGCTGGATCGGCTTGCATCTGCC
                   CAGAATTTCGTCACTACCTTGATGGCATCGAACGTGT
                   GGATTCCTTATCGCTGTCTCCCCACAAATGGCTTTTA
                   CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAACC
                   GGATGTATCTAGTTTACATGTCGAGGAGGTCAACAAG
                   AAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTAT
                   ATATGACGCATACAATTGTTGGCGGAATCTATATGTT
                   ACGTTTGGCTGTAGGTAGCAGCTTGACAGAGGAACA
                   TCACGTGCGCCGCGTTTGGGACTTGATCCAGAAGCTT
                   ACGGACGACCTGCTTAAAGAGGCGTGA
Tdc (tdc from      ATGGGTTCTATTGACTCGACGAATGTGGCCATGTCTA
C. roseus)         ATTCTCCTGTTGGCGAGTTTAAGCCCCTTGAAGCAGA
SEQ ID NO: 260     AGAGTTCCGTAAACAGGCACACCGCATGGTGGATTTT
                   ATTGCGGATTATTACAAGAACGTAGAAACATACCCG
                   GTCCTTTCCGAGGTTGAACCCGGCTATCTGCGCAAAC
                   GTATTCCCGAAACCGCACCATACCTGCCGGAGCCACT
                   TGATGATATTATGAAGGATATTCAAAAGGACATTATC
                   CCCGGAATGACGAACTGGATGTCCCCGAACTTTTACG
                   CCTTCTTCCCGGCCACAGTTAGCTCAGCAGCTTTCTTG
                   GGGGAAATGCTTTCAACGGCCCTTAACAGCGTAGGA
                   TTTACCTGGGTCAGTTCCCCGGCAGCGACTGAATTAG
                   AGATGATCGTTATGGATTGGCTTGCGCAAATTTTGAA
                   ACTTCCAAAAAGCTTTATGTTCTCCGGAACCGGGGGT
                   GGTGTCATCCAAAACACTACGTCAGAGTCGATCTTGT
                   GCACTATTATCGCGGCCCGTGAACGCGCCTTGGAAAA
                   ATTGGGCCCTGATTCAATTGGTAAGCTTGTCTGCTAT
                   GGGTCCGATCAAACGCACACAATGTTTCCGAAAACCT
                   GTAAGTTAGCAGGAATTTATCCGAATAATATCCGCCT
                   TATCCCTACCACGGTAGAAACCGACTTTGGCATCTCA
                   CCGCAGGTACTTCGCAAGATGGTCGAAGACGACGTC
                   GCTGCGGGGTACGTTCCCTTATTTTTGTGTGCCACCTT
                   GGGAACGACATCAACTACGGCAACAGATCCTGTAGA
                   TTCGCTGTCCGAAATCGCAAACGAGTTTGGTATCTGG
                   ATTCATGTCGACGCCGCATATGCTGGATCGGCTTGCA
                   TCTGCCCAGAATTTCGTCACTACCTTGATGGCATCGA
                   ACGTGTGGATTCCTTATCGCTGTCTCCCCACAAATGG
                   CTTTTAGCATATCTGGATTGCACGTGCTTGTGGGTAA
                   AACAACCTCACCTGCTGCTTCGCGCTTTAACGACTAA
                   TCCCGAATACTTGAAGAATAAACAGAGTGATTTAGAT
                   AAGGTCGTGGATTTTAAGAACTGGCAGATCGCAACA
                   GGACGTAAGTTCCGCTCTTTAAAACTTTGGTTAATTC
                   TGCGTTCCTACGGGGTAGTTAACCTGCAAAGTCATAT
                   CCGTAGTGATGTAGCGATGGGGAAGATGTTTGAGGA
                   ATGGGTCCGTTCCGATAGCCGCTTTGAAATCGTCGTG
                   CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAACC
                   GGATGTATCTAGTTTACATGTCGAGGAGGTCAACAAG
                   AAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTAT
                   ATATGACGCATACAATTGTTGGCGGAATCTATATGTT
                   ACGTTTGGCTGTAGGTAGCAGCTTGACAGAGGAACA
                   TCACGTGCGCCGCGTTTGGGACTTGATCCAGAAGCTT
                   ACGGACGACCTGCTTAAAGAGGCGTGA
fbrAroG-Tdc (tdc   ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga
from Clostridium   tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg
sporogenes); RBS   ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg
and leader region  aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc
underlined         ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg
SEQ ID NO: 261     aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg
                   attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc
                   aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg
                   atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac
                   cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat
                   ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca
                   ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg
                   gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt
                   gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat
                   ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg
                   ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg
                   gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc
                   accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca
                   gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGAAATT
                   TTGGCGCAAGTATACGCAACAGGAGATGGATGAGAA
                   AATCACAGAATCGCTTGAGAAGACATTAAATTACGA
                   TAACACGAAAACCATCGGCATCCCAGGTACTAAGCT
                   GGATGATACTGTATTTTATGACGATCACTCCTTCGTT
                   AAGCACTCTCCCTATTTACGTACGTTCATCCAAAACC
                   CTAATCACATTGGTTGTCACACGTACGATAAAGCAGA
                   CATCTTGTTTGGCGGCACGTTTGACATCGAACGCGAA
                   CTGATTCAGCTTTTGGCCATCGATGTCTTAAACGGAA
                   ATGATGAGGAATTCGATGGATATGTGACACAGGGGG
                   GAACCGAGGCGAATATTCAGGCAATGTGGGTTTATC
                   GTAACTATTTCAAAAAAGAACGTAAAGCAAAACATG
                   AGGAAATCGCAATCATCACGAGCGCGGATACCCATT
                   ACAGTGCATATAAGGGGAGCGACTTGCTGAACATTG
                   ATATTATCAAGGTCCCAGTAGACTTCTATTCGCGTAA
                   GATCCAGGAGAACACGTTAGACTCGATTGTCAAGGA
                   GGCGAAGGAAATTGGAAAGAAGTACTTCATTGTCAT
                   CTCAAACATGGGTACGACTATGTTTGGCAGTGTAGAC
                   GACCCTGATCTTTATGCTAACATTTTTGATAAGTATA
                   ACTTAGAATACAAAATCCACGTCGATGGAGCTTTTGG
                   GGGTTTCATTTATCCTATCGATAATAAGGAGTGCAAA
                   ACAGATTTCTCGAACAAGAACGTCTCATCCATCACGC
                   TTGACGGTCACAAAATGCTTCAAGCCCCCTATGGGAC
                   TGGTATCTTCGTGTCACGTAAGAACTTGATCCATAAC
                   ACCCTGACAAAGGAAGCAACGTATATTGAAAACCTG
                   GACGTTACCCTGAGTGGGTCCCGCTCCGGATCCAACG
                   CCGTTGCGATCTGGATGGTTTTAGCCTCTTATGGCCC
                   CTACGGGTGGATGGAGAAGATTAACAAGTTGCGCAA
                   TCGCACTAAGTGGCTTTGCAAGCAGCTTAACGACATG
                   CGCATCAAATACTATAAGGAGGATAGCATGAATATC
                   GTCACGATTGAAGAGCAATACGTAAATAAAGAGATT
                   GCAGAGAAATACTTCCTTGTGCCTGAAGTACACAATC
                   CTACCAACAATTGGTACAAGATTGTAGTCATGGAACA
                   TGTTGAACTTGACATCTTGAACTCCCTTGTTTATGATT
                   TACGTAAATTCAACAAGGAGCACCTGAAGGCAATGT
                   GA
Tdc (tdc from      ATGAAATTTTGGCGCAAGTATACGCAACAGGAGATG
Clostridium        GATGAGAAAATCACAGAATCGCTTGAGAAGACATTA
sporogenes)        AATTACGATAACACGAAAACCATCGGCATCCCAGGT
SEQ ID NO: 262     ACTAAGCTGGATGATACTGTATTTTATGACGATCACT
                   CCTTCGTTAAGCACTCTCCCTATTTACGTACGTTCATC
                   CAAAACCCTAATCACATTGGTTGTCACACGTACGATA
                   AAGCAGACATCTTGTTTGGCGGCACGTTTGACATCGA
                   ACGCGAACTGATTCAGCTTTTGGCCATCGATGTCTTA
                   AACGGAAATGATGAGGAATTCGATGGATATGTGACA
                   CAGGGGGGAACCGAGGCGAATATTCAGGCAATGTGG
                   GTTTATCGTAACTATTTCAAAAAAGAACGTAAAGCAA
                   AACATGAGGAAATCGCAATCATCACGAGCGCGGATA
                   CCCATTACAGTGCATATAAGGGGAGCGACTTGCTGA
                   ACATTGATATTATCAAGGTCCCAGTAGACTTCTATTC
                   GCGTAAGATCCAGGAGAACACGTTAGACTCGATTGT
                   CAAGGAGGCGAAGGAAATTGGAAAGAAGTACTTCAT
                   TGTCATCTCAAACATGGGTACGACTATGTTTGGCAGT
                   GTAGACGACCCTGATCTTTATGCTAACATTTTTGATA
                   AGTATAACTTAGAATACAAAATCCACGTCGATGGAG
                   CTTTTGGGGGTTTCATTTATCCTATCGATAATAAGGA
                   GTGCAAAACAGATTTCTCGAACAAGAACGTCTCATCC
                   ATCACGCTTGACGGTCACAAAATGCTTCAAGCCCCCT
                   ATGGGACTGGTATCTTCGTGTCACGTAAGAACTTGAT
                   CCATAACACCCTGACAAAGGAAGCAACGTATATTGA
                   AAACCTGGACGTTACCCTGAGTGGGTCCCGCTCCGGA
                   TCCAACGCCGTTGCGATCTGGATGGTTTTAGCCTCTT
                   ATGGCCCCTACGGGTGGATGGAGAAGATTAACAAGT
                   TGCGCAATCGCACTAAGTGGCTTTGCAAGCAGCTTAA
                   CGACATGCGCATCAAATACTATAAGGAGGATAGCAT
                   GAATATCGTCACGATTGAAGAGCAATACGTAAATAA
                   AGAGATTGCAGAGAAATACTTCCTTGTGCCTGAAGTA
                   CACAATCCTACCAACAATTGGTACAAGATTGTAGTCA
                   TGGAACATGTTGAACTTGACATCTTGAACTCCCTTGT
                   TTATGATTTACGTAAATTCAACAAGGAGCACCTGAAG
                   GCAATGTGA
fbrArG-trpDH-      Ctctagaaataattttgtttaactttaagaaggagatatacat
ipdC-iad1 (RBS     atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg
and leader region  cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga
underlined)        aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca
SEQ ID NO: 263     tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg
                   aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta
                   cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac
                   gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg
                   gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc
                   gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc
                   ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta
                   atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt
                   gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa
                   ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc
                   agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat
                   ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt
                   gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct
                   ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt
                   acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaaggaga
                   tatacatATGCTGTTATTCGAGACTGTGCGTGAAATGGGT
                   CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG
                   AGATCAAGGCAATTATCGCAATCCACGATACCACCTT
                   AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT
                   ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC
                   TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA
                   ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC
                   TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC
                   ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC
                   ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC
                   GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG
                   GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA
                   CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT
                   AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT
                   GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA
                   AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA
                   CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG
                   AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC
                   GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC
                   CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT
                   CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG
                   AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG
                   ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT
                   ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA
                   CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA
                   ACAAGTTCATAACATCTACGATACGTTATTAGCGATT
                   TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC
                   GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC
                   AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG
                   TGAAATGtaagaaggagatatacatATGCGTACACCCTACTGTG
                   TCGCCGATTATCTTTTAGATCGTCTGACGGACTGCGG
                   GGCCGATCACCTGTTTGGCGTACCGGGCGATTACAAC
                   TTGCAGTTTCTGGACCACGTCATTGACTCACCAGATA
                   TCTGCTGGGTAGGGTGTGCGAACGAGCTTAACGCGA
                   GCTACGCTGCTGACGGATATGCGCGTTGTAAAGGCTT
                   TGCTGCACTTCTTACTACCTTCGGGGTCGGTGAGTTA
                   TCGGCGATGAACGGTATCGCAGGCTCGTACGCTGAG
                   CACGTCCCGGTATTACACATTGTGGGAGCTCCGGGTA
                   CCGCAGCTCAACAGCGCGGAGAACTGTTACACCACA
                   CGCTGGGCGACGGAGAATTCCGCCACTTTTACCATAT
                   GTCCGAGCCAATTACTGTAGCCCAGGCTGTACTTACA
                   GAGCAAAATGCCTGTTACGAGATCGACCGTGTTTTGA
                   CCACGATGCTTCGCGAGCGCCGTCCCGGGTATTTGAT
                   GCTGCCAGCCGATGTTGCCAAAAAAGCTGCGACGCC
                   CCCAGTGAATGCCCTGACGCATAAACAAGCTCATGCC
                   GATTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTG
                   AAAATAAATTAGCCATGTCGAAACGCACCGCCTTGTT
                   GGCGGACTTTCTGGTCCTGCGCCATGGCCTTAAACAC
                   GCCCTTCAGAAATGGGTCAAAGAAGTCCCGATGGCC
                   CACGCTACGATGCTTATGGGTAAGGGGATTTTTGATG
                   AACGTCAAGCGGGATTTTATGGAACTTATTCCGGTTC
                   GGCGAGTACGGGGGCGGTAAAGGAAGCGATTGAGGG
                   AGCCGACACAGTTCTTTGCGTGGGGACACGTTTCACC
                   GATACACTGACCGCTGGATTCACACACCAACTTACTC
                   CGGCACAAACGATTGAGGTGCAACCCCATGCGGCTC
                   GCGTGGGGGATGTATGGTTTACGGGCATTCCAATGAA
                   TCAAGCCATTGAGACTCTTGTCGAGCTGTGCAAACAG
                   CACGTCCACGCAGGACTGATGAGTTCGAGCTCTGGG
                   GCGATTCCTTTTCCACAACCAGATGGTAGTTTAACTC
                   AAGAAAACTTCTGGCGCACATTGCAAACCTTTATCCG
                   CCCAGGTGATATCATCTTAGCAGACCAGGGTACTTCA
                   GCCTTTGGAGCAATTGACCTGCGCTTACCAGCAGACG
                   TGAACTTTATTGTGCAGCCGCTGTGGGGGTCTATTGG
                   TTATACTTTAGCTGCGGCCTTCGGAGCGCAGACAGCG
                   TGTCCAAACCGTCGTGTGATCGTATTGACAGGAGATG
                   GAGCAGCGCAGTTGACCATTCAGGAGTTAGGCTCGA
                   TGTTACGCGATAAGCAGCACCCCATTATCCTGGTCCT
                   GAACAATGAGGGGTATACAGTTGAACGCGCCATTCA
                   TGGTGCGGAACAACGCTACAATGACATCGCTTTATGG
                   AATTGGACGCACATCCCCCAAGCCTTATCGTTAGATC
                   CCCAATCGGAATGTTGGCGTGTGTCTGAAGCAGAGC
                   AACTGGCTGATGTTCTGGAAAAAGTTGCTCATCATGA
                   ACGCCTGTCGTTGATCGAGGTAATGTTGCCCAAGGCC
                   GATATCCCTCCGTTACTGGGAGCCTTGACCAAGGCTT
                   TAGAAGCCTGCAACAACGCTTAAAGGTtaagaaggagatata
                   catATGCCCACCTTGAACTTGGACTTACCCAACGGTAT
                   TAAGAGCACGATTCAGGCAGACCTTTTCATCAATAAT
                   AAGTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAA
                   CTATTAATCCGTCTACGGGGAAAGAGATCGGACAGG
                   TGGCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAG
                   TTAAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGG
                   GGAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTG
                   ATTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATG
                   AGTTAGCGGCAATTGAATCACTGGACAATGGGAAAG
                   CGTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGT
                   GGCCGCAAACTTACGTTACTACGGCGGTTGGGCTGAT
                   AAAAACCACGGTAAAGTCATGGAGGTAGACACAAAG
                   CGCCTGAACTATACCCGCCACGAGCCGATCGGGGTTT
                   GCGGACAAATCATTCCGTGGAATTTCCCGCTTTTGAT
                   GTTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGG
                   AACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCT
                   TAAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGC
                   CGGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGA
                   TTCGGACCGGTGGCGGGGGCCGCGATCTCGCAACAC
                   ATGGACATCGATAAGATTGCCTTTACAGGATCGACAT
                   TGGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGAC
                   TAACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAA
                   ATCCCCGAATATCATTTTCAAAGATGCCGACCTTGAC
                   CAAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTA
                   ACCACGGACAATGCTGCTGCGCTGGATCGCGCGTATA
                   TGTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA
                   ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC
                   CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC
                   GCAGTTACAATACGACCGTATCATGGAATACATCGA
                   ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG
                   CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC
                   AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT
                   GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT
                   CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG
                   CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT
                   TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC
                   AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT
                   AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA
                   AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC
                   CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT
                   AACCTTTCTCAACCGGCGCCCATTTGA
trpDH              ATGCTGTTATTCGAGACTGTGCGTGAAATGGGTCATG
SEQ ID NO: 264     AGCAAGTCCTTTTCTGTCATAGCAAGAATCCCGAGAT
                   CAAGGCAATTATCGCAATCCACGATACCACCTTAGGA
                   CCGGCTATGGGCGCAACTCGTATCTTACCTTATATTA
                   ATGAGGAGGCTGCCCTGAAAGATGCATTACGTCTGTC
                   CCGCGGAATGACTTACAAAGCAGCCTGCGCCAATATT
                   CCCGCCGGGGGCGGCAAAGCCGTCATCATCGCTAAC
                   CCCGAAAACAAGACCGATGACCTGTTACGCGCATAC
                   GGCCGTTTCGTGGACAGCTTGAACGGCCGTTTCATCA
                   CCGGGCAGGACGTTAACATTACGCCCGACGACGTTC
                   GCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGT
                   CTCAGAAAAGTCGGGAGGGCCGGCACCTATCACCTC
                   TCTGGGAGTATTTTTAGGCATCAAAGCCGCTGTAGAG
                   TCGCGTTGGCAGTCTAAACGCCTGGATGGCATGAAA
                   GTGGCGGTGCAAGGACTTGGGAACGTAGGAAAAAAT
                   CTTTGTCGCCATCTGCATGAACACGATGTACAACTTT
                   TTGTGTCTGATGTCGATCCAATCAAGGCCGAGGAAGT
                   AAAACGCTTATTCGGGGCGACTGTTGTCGAACCGACT
                   GAAATCTATTCTTTAGATGTTGATATTTTTGCACCGTG
                   TGCACTTGGGGGTATTTTGAATAGCCATACCATCCCG
                   TTCTTACAAGCCTCAATCATCGCAGGAGCAGCGAATA
                   ACCAGCTGGAGAACGAGCAACTTCATTCGCAGATGC
                   TTGCGAAAAAGGGTATTCTTTACTCACCAGACTACGT
                   TATCAATGCAGGAGGACTTATCAATGTTTATAACGAA
                   ATGATCGGATATGACGAGGAAAAAGCATTCAAACAA
                   GTTCATAACATCTACGATACGTTATTAGCGATTTTCG
                   AAATTGCAAAAGAACAAGGTGTAACCACCAACGACG
                   CGGCCCGTCGTTTAGCAGAGGATCGTATCAACAACTC
                   CAAACGCTCAAAGAGTAAAGCGATTGCGGCGTGA
ipdC               ATGCGTACACCCTACTGTGTCGCCGATTATCTTTTAG
SEQ ID NO: 265     ATCGTCTGACGGACTGCGGGGCCGATCACCTGTTTGG
                   CGTACCGGGCGATTACAACTTGCAGTTTCTGGACCAC
                   GTCATTGACTCACCAGATATCTGCTGGGTAGGGTGTG
                   CGAACGAGCTTAACGCGAGCTACGCTGCTGACGGAT
                   ATGCGCGTTGTAAAGGCTTTGCTGCACTTCTTACTAC
                   CTTCGGGGTCGGTGAGTTATCGGCGATGAACGGTATC
                   GCAGGCTCGTACGCTGAGCACGTCCCGGTATTACACA
                   TTGTGGGAGCTCCGGGTACCGCAGCTCAACAGCGCG
                   GAGAACTGTTACACCACACGCTGGGCGACGGAGAAT
                   TCCGCCACTTTTACCATATGTCCGAGCCAATTACTGT
                   AGCCCAGGCTGTACTTACAGAGCAAAATGCCTGTTAC
                   GAGATCGACCGTGTTTTGACCACGATGCTTCGCGAGC
                   GCCGTCCCGGGTATTTGATGCTGCCAGCCGATGTTGC
                   CAAAAAAGCTGCGACGCCCCCAGTGAATGCCCTGAC
                   GCATAAACAAGCTCATGCCGATTCCGCCTGTTTAAAG
                   GCTTTTCGCGATGCAGCTGAAAATAAATTAGCCATGT
                   CGAAACGCACCGCCTTGTTGGCGGACTTTCTGGTCCT
                   GCGCCATGGCCTTAAACACGCCCTTCAGAAATGGGTC
                   AAAGAAGTCCCGATGGCCCACGCTACGATGCTTATG
                   GGTAAGGGGATTTTTGATGAACGTCAAGCGGGATTTT
                   ATGGAACTTATTCCGGTTCGGCGAGTACGGGGGCGGT
                   AAAGGAAGCGATTGAGGGAGCCGACACAGTTCTTTG
                   CGTGGGGACACGTTTCACCGATACACTGACCGCTGGA
                   TTCACACACCAACTTACTCCGGCACAAACGATTGAGG
                   TGCAACCCCATGCGGCTCGCGTGGGGGATGTATGGTT
                   TACGGGCATTCCAATGAATCAAGCCATTGAGACTCTT
                   GTCGAGCTGTGCAAACAGCACGTCCACGCAGGACTG
                   ATGAGTTCGAGCTCTGGGGCGATTCCTTTTCCACAAC
                   CAGATGGTAGTTTAACTCAAGAAAACTTCTGGCGCAC
                   ATTGCAAACCTTTATCCGCCCAGGTGATATCATCTTA
                   GCAGACCAGGGTACTTCAGCCTTTGGAGCAATTGACC
                   TGCGCTTACCAGCAGACGTGAACTTTATTGTGCAGCC
                   GCTGTGGGGGTCTATTGGTTATACTTTAGCTGCGGCC
                   TTCGGAGCGCAGACAGCGTGTCCAAACCGTCGTGTG
                   ATCGTATTGACAGGAGATGGAGCAGCGCAGTTGACC
                   ATTCAGGAGTTAGGCTCGATGTTACGCGATAAGCAGC
                   ACCCCATTATCCTGGTCCTGAACAATGAGGGGTATAC
                   AGTTGAACGCGCCATTCATGGTGCGGAACAACGCTA
                   CAATGACATCGCTTTATGGAATTGGACGCACATCCCC
                   CAAGCCTTATCGTTAGATCCCCAATCGGAATGTTGGC
                   GTGTGTCTGAAGCAGAGCAACTGGCTGATGTTCTGGA
                   AAAAGTTGCTCATCATGAACGCCTGTCGTTGATCGAG
                   GTAATGTTGCCCAAGGCCGATATCCCTCCGTTACTGG
                   GAGCCTTGACCAAGGCTTTAGAAGCCTGCAACAACG
                   CTTAA
Iad1               ATGCCCACCTTGAACTTGGACTTACCCAACGGTATTA
SEQ ID NO: 266     AGAGCACGATTCAGGCAGACCTTTTCATCAATAATAA
                   GTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAACT
                   ATTAATCCGTCTACGGGGAAAGAGATCGGACAGGTG
                   GCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAGTT
                   AAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGGG
                   GAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTGA
                   TTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATGA
                   GTTAGCGGCAATTGAATCACTGGACAATGGGAAAGC
                   GTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGTG
                   GCCGCAAACTTACGTTACTACGGCGGTTGGGCTGATA
                   AAAACCACGGTAAAGTCATGGAGGTAGACACAAAGC
                   GCCTGAACTATACCCGCCACGAGCCGATCGGGGTTTG
                   CGGACAAATCATTCCGTGGAATTTCCCGCTTTTGATG
                   TTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGGA
                   ACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCTT
                   AAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGCC
                   GGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGAT
                   TCGGACCGGTGGCGGGGGCCGCGATCTCGCAACACA
                   TGGACATCGATAAGATTGCCTTTACAGGATCGACATT
                   GGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGACT
                   AACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAAA
                   TCCCCGAATATCATTTTCAAAGATGCCGACCTTGACC
                   AAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTAA
                   CCACGGACAATGCTGCTGCGCTGGATCGCGCGTATAT
                   GTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA
                   ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC
                   CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC
                   GCAGTTACAATACGACCGTATCATGGAATACATCGA
                   ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG
                   CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC
                   AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT
                   GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT
                   CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG
                   CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT
                   TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC
                   AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT
                   AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA
                   AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC
                   CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT
                   AACCTTTCTCAACCGGCGCCCATTTGA
TrpEDCBA (RBS      Ctctagaaataattttgtttaactttaagaaggagatatacat
and leader region  atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa
underlined)        cccgactgcgctattcaccagagtgtggggatcgtccggcaacgctgctgctggaatc
SEQ ID NO: 267     cgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc
                   gcattacagcattaagtgacactgtcacaatccaggcgctaccggcaatggagaagcc
                   ctgagacactactggataacgccagcctgcgggtgtggaaaatgaacaatcaccaaac
                   tgccgcgtactgcgcacccgcctgtcagtccactgctggatgaagacgcccgcttatgc
                   tcccatcggtattgacgctaccgcttattacagaatctgagaatgtaccgaaggaagaa
                   cgagaagcaatgacttcggcggcctgactcttatgaccagtggcgggatttgaaaattt
                   accgcaactgtcagcggaaaatagctgccctgatactgatttatctcgctgaaacgctga
                   tggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgatgctccgaatg
                   aagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccga
                   agccgcgccgccgctgccggtggtaccgtgccgcatatgcgagtgaatgtaaccaga
                   gcgatgaagagacggtggtgtagtgcgatgagcaaaaagcgattcgcgccggagaa
                   attaccaggtggtgccatctcgccgtactctctgccctgcccgtcaccgctggcagccta
                   ttacgtgctgaaaaagagtaatcccagcccgtacatgatatatgcaggataatgatacac
                   cctgtaggcgcgtcgccggaaagacgctcaagtatgacgccaccagccgccagattg
                   agatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggacgctggac
                   agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctactga
                   acatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggca
                   gccgctacgtcgccgatctcaccaaagagaccgttactcttacgtgatgcacctagtctc
                   ccgcgagaggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtat
                   gaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaag
                   cagaaggtcgtcgacgcggcagctacggcggcgcggtaggttatataccgcgcatgg
                   cgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccg
                   tgcaagccggtgctggcgtagtccagattctgaccgcagtcggaagccgacgaaactc
                   gtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagac
                   gactaatggctgacattctgctgctcgataatatcgactcattacgtacaacctggcagat
                   cagagcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagacc
                   ttaattgaacgcctggcgacgatgagcaatccggtgctgatgctactcctggccccggt
                   gtgccgagcgaagccggagtatgccggaactcctcacccgcttgcgtggcaagctgc
                   caattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcgg
                   tcaggcgggcgaaattcacacggtaaagcgtcgagcattgaacatgacggtcaggcg
                   atgatgccggattaacaaacccgctgccagtggcgcgttatcactcgctggaggcagt
                   aacattccggccggataaccatcaacgcccatataatggcatggtgatggcggtgcgtc
                   acgatgcagatcgcgtagtggattccagaccatccggaatccattcttactacccaggg
                   cgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa
                   cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaa
                   agccaccagctgattcagcggtggtacgtggcgagctgaagccggaacaactggcgg
                   cggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcag
                   caaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgatgccgat
                   atcgtcggtactggcggtgacggcagcaacagcatcaatatactaccgccagtgcgat
                   gtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagt
                   aaatccggctcgtcggatctgctggcggcgacggtattaatcagatatgaacgccgata
                   aatcgcgccaggcgctggatgagttaggcgtctgatcctcatgcgccgaagtatcaca
                   ccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaac
                   gtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtc
                   cggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggc
                   agtggtgcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccg
                   aactacatgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacac
                   cctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaac
                   acgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtc
                   gccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttctt
                   gaggtactgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggt
                   aaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacc
                   cgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga
                   catttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgt
                   cgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttata
                   aacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatt
                   tcctccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgat
                   ccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagta
                   ctggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgt
                   gctgaccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaa
                   ggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg
                   cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaat
                   acttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgtt
                   gatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaag
                   tatgtggcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttac
                   ggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcaggaa
                   gtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatg
                   tggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagat
                   cagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggc
                   tttaagtgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcg
                   acaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcg
                   cttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcac
                   aaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaag
                   acgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaat
                   gacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctga
                   tgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttc
                   aggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaa
                   atgccagaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagatttg
                   ctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc
                   ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtc
                   ggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacg
                   ttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccg
                   gtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactggtc
                   cggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc
                   cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctg
                   gaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc
                   catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcct
                   ggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtg
                   ggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaaga
                   gtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctca
                   acagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagccttta
                   aaacgctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcc
                   catgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactggtggttaacct
                   ttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagggga
                   aatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgc
                   attcgttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgata
                   cgctaattgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactg
                   gcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccc
                   cggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatc
                   ggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtg
                   cgagaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcg
                   cccttccgccaggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatg
                   ccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcg
                   cgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttg
                   cgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccg
                   gatcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccat
                   cgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaag
                   cttttgtacaaccgatgaaagcggcgacgcgcagtta
trpE               atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa
SEQ ID NO: 268     cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaatc
                   cgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc
                   gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagcc
                   ctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaac
                   tgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgc
                   tccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaa
                   cgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaattt
                   accgcaactgtcagcggaaaatagctgccctgatttctgatttatctcgctgaaacgctga
                   tggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatg
                   aagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccga
                   agccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccaga
                   gcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaa
                   attttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagccta
                   ttacgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggataatgatttcac
                   cctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattg
                   agatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggac
                   agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctga
                   acatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggca
                   gccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctc
                   ccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtat
                   gaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaag
                   cagaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatgg
                   cgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccg
                   tgcaagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactc
                   gtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagac
                   gttcta
trpD               atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagtt
SEQ ID NO: 269     gcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagaccttaat
                   tgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggtgtgc
                   cgagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaatt
                   attggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcag
                   gcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtt
                   tgccggattaacaaacccgctgccagtggcgcgttatcactcgctggttggcagtaacat
                   tccggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgcgtcacga
                   tgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccagggcgctc
                   gcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacg
                   ctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagcc
                   accagctgttttcagcggtggtacgtggcgagctgaagccggaacaactggcggcggc
                   gctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcagcaac
                   cgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgt
                   cggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgc
                   cgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatc
                   cggctcgtcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcg
                   cgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccgga
                   ttccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctg
                   ggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtccggaa
                   ctggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggt
                   gcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactac
                   atgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacaccctacc
                   accaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacacgctt
                   gttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccat
                   gttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggt
                   actgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa
trpC               atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaa
SEQ ID NO: 270     agagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcgacattttt
                   atgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgt
                   caaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttataaacatta
                   cgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccc
                   catcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttacc
                   agatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggat
                   gacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctga
                   ccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgt
                   tggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagct
                   tgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttac
                   gctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatgg
                   cccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtg
                   gcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgg
                   gttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcaggaagtgatg
                   gctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcg
                   gacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagct
                   gtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag
                   tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaac
                   ggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcgcttgg
                   caacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaac
                   cggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgc
                   acgtcttttggcctcggttttccagacgctgcgcgcatattaa
trpB               atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcct
SEQ ID NO: 271     gatgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatt
                   tcaggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgacca
                   aatgccagaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagattt
                   gctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaag
                   cggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtc
                   ggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacg
                   ttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccg
                   gtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactggtc
                   cggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc
                   cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctg
                   gaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc
                   catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcct
                   ggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtg
                   ggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaaga
                   gtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctca
                   acagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagccttta
                   aaacgctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcc
                   catgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactggtggttaacct
                   ttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagggga
                   aatctg
trpA               atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgtt
SEQ ID NO: 272     cctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaat
                   tgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactggcggatg
                   gcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccccggcgca
                   gtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggccttttg
                   atgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaag
                   tcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgc
                   caggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatg
                   atttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcgcgagcggg
                   cgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctg
                   aaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggta
                   aaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaat
                   catcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtaca
                   accgatgaaagcggcgacgcgcagttaa
fbrS40FTrpE-       ctctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccga
DCBA (leader       ctctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcacc
region and RBS     agttgtgtggggatcgtccggcaacgctgctgctggaattcgcagatatcgacagcaaa
underlined)        gatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgac
SEQ ID NO: 273     actgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactggataac
                   gccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttccc
                   gcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgcttt
                   ccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcgg
                   cggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaa
                   aatagctgccctgatttctgatttatctcgctgaaacgctgatggtgattgaccatcagaaa
                   aaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctc
                   actgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccg
                   gtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggt
                   gtagtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctc
                   gccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgctgaaaaagagta
                   atcccagcccgtacatgattttatgcaggataatgatttcaccctgtttggcgcgtcgccg
                   gaaagttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccgg
                   aacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc
                   atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatc
                   tcgcccgtaatgacctggcacgcatttgcacacccggcagccgctacgtcgccgatctc
                   accaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagctgc
                   gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagc
                   ggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcg
                   gcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcatt
                   gtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcg
                   tagtccttgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctg
                   tactgcgcgctattgccaccgcgcatcatgcacaggagacgttctaatggctgacattct
                   gctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggt
                   cataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcg
                   acgatgagcaatccggtgctgatgctttctcctggccccggtgtgccgagcgaagccgg
                   ttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgcctc
                   ggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattct
                   tcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaa
                   acccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaa
                   ccatcaacgcccattttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgttt
                   gtggattccagttccatccggaatccattcttactacccagggcgctcgcctgctggaac
                   aaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattc
                   tggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttttca
                   gcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcat
                   gaaaattcgcggtgaacacccgaacgagatcgccggggcagcaaccgcgctactgga
                   aaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggtactggcgg
                   tgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcg
                   ggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcgga
                   tctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgct
                   ggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcg
                   atgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctgggaccattgatt
                   aacccggcgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgcc
                   gattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggc
                   gggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcga
                   aattaagagctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagca
                   attggcaggcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggta
                   aaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcct
                   gcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtg
                   gttccgcttacgacagagtcaccgcactggcggcacgagggtaaatgatgcaaaccgtt
                   ttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaac
                   cgctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttca
                   gggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtg
                   atccgtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaat
                   ttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccccatcgtcagcc
                   aaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg
                   gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaat
                   atcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagt
                   aatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaaca
                   accgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaac
                   tggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggtgcgc
                   gagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgattt
                   gaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtg
                   ggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgc
                   gacatcaccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccg
                   ttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggacaaagctaag
                   gtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaat
                   ctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaact
                   cttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggg
                   agcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctgg
                   cggggggcttaggcgcagataactgcgtggaagcggcacaaaccggctgcgccggg
                   cttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctc
                   ggttttccagacgctgcgcgcatattaaggaaaggaacaatgacaacattacttaacccc
                   tattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgccagct
                   ggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacct
                   gctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagcc
                   gggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcggcgcgcata
                   aaactaaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaatt
                   attgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgc
                   tcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaac
                   gttttccggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgacc
                   ctgaaagatgcctgtaatgaggcgctacgcgactggtccggcagttatgaaaccgcgc
                   actatatgctgggtaccgcagctggcccgcatccttacccgaccattgtgcgtgagtttca
                   gcggatgattggcgaagaaacgaaagcgcagattctggaaagagaaggtcgcctgcc
                   ggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttca
                   tcaacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaac
                   tggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctatttcggtatgaaag
                   cgccgatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccggg
                   ctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgat
                   tacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaag
                   ggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcg
                   cgaaaatccggaaaaagagcagctactggtggttaacctttccggtcgcggcgataaag
                   acatcttcaccgttcacgatattttgaaagcacgaggggaaatctgatggaacgctacga
                   atctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc
                   ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgct
                   gacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattca
                   aaacgccacactgcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgc
                   tggcactcattcgccagaagcacccgaccattcccatcggccttttgatgtatgccaacct
                   ggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcgattc
                   ggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttg
                   cgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgcca
                   gatagcctcttacggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgc
                   agaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacg
                   ctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccgcgattg
                   atgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacata
                   ttaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcg
                   gcgacgcgcagttaa
fbrTrpE            atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa
SEQ ID NO: 274     cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc
                   gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg
                   cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct
                   gttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactg
                   ccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctc
                   cctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacg
                   agaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttac
                   cgcaactgtcagcggaaaatagctgccctgatttctgatttatctcgctgaaacgctgatg
                   gtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaa
                   gaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaag
                   ccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagc
                   gatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaatt
                   ttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctatta
                   cgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggataatgatttcaccc
                   tgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgag
                   atttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacag
                   agacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctgaac
                   atctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagc
                   cgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctccc
                   gcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga
                   atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagca
                   gaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcg
                   atctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtg
                   caagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactcgt
                   aataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagacgtt
                   cta
trpDH-             ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga
fldABCDacuIfldH    tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg
(leader region and ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg
RBS underlined)    aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc
SEQ ID NO: 275     ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg
                   aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg
                   attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc
                   aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg
                   atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac
                   cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat
                   ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca
                   ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg
                   gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt
                   gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat
                   ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg
                   ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg
                   gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc
                   accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca
                   gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGCTGTT
                   ATTCGAGACTGTGCGTGAAATGGGTCATGAGCAAGT
                   CCTTTTCTGTCATAGCAAGAATCCCGAGATCAAGGCA
                   ATTATCGCAATCCACGATACCACCTTAGGACCGGCTA
                   TGGGCGCAACTCGTATCTTACCTTATATTAATGAGGA
                   GGCTGCCCTGAAAGATGCATTACGTCTGTCCCGCGGA
                   ATGACTTACAAAGCAGCCTGCGCCAATATTCCCGCCG
                   GGGGCGGCAAAGCCGTCATCATCGCTAACCCCGAAA
                   ACAAGACCGATGACCTGTTACGCGCATACGGCCGTTT
                   CGTGGACAGCTTGAACGGCCGTTTCATCACCGGGCAG
                   GACGTTAACATTACGCCCGACGACGTTCGCACTATTT
                   CGCAGGAGACTAAGTACGTGGTAGGCGTCTCAGAAA
                   AGTCGGGAGGGCCGGCACCTATCACCTCTCTGGGAGT
                   ATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGG
                   CAGTCTAAACGCCTGGATGGCATGAAAGTGGCGGTG
                   CAAGGACTTGGGAACGTAGGAAAAAATCTTTGTCGC
                   CATCTGCATGAACACGATGTACAACTTTTTGTGTCTG
                   ATGTCGATCCAATCAAGGCCGAGGAAGTAAAACGCT
                   TATTCGGGGCGACTGTTGTCGAACCGACTGAAATCTA
                   TTCTTTAGATGTTGATATTTTTGCACCGTGTGCACTTG
                   GGGGTATTTTGAATAGCCATACCATCCCGTTCTTACA
                   AGCCTCAATCATCGCAGGAGCAGCGAATAACCAGCT
                   GGAGAACGAGCAACTTCATTCGCAGATGCTTGCGAA
                   AAAGGGTATTCTTTACTCACCAGACTACGTTATCAAT
                   GCAGGAGGACTTATCAATGTTTATAACGAAATGATCG
                   GATATGACGAGGAAAAAGCATTCAAACAAGTTCATA
                   ACATCTACGATACGTTATTAGCGATTTTCGAAATTGC
                   AAAAGAACAAGGTGTAACCACCAACGACGCGGCCCG
                   TCGTTTAGCAGAGGATCGTATCAACAACTCCAAACGC
                   TCAAAGAGTAAAGCGATTGCGGCGTGAAATGtaagaagg
                   agatatacatATGGAAAACAACACCAATATGTTCTCTGGAG
                   TGAAGGTGATCGAACTGGCCAACTTTATCGCTGCTCC
                   GGCGGCAGGTCGCTTCTTTGCTGATGGGGGAGCAGA
                   AGTAATTAAGATCGAATCTCCAGCAGGCGACCCGCT
                   GCGCTACACGGCCCCATCAGAAGGACGCCCGCTTTCT
                   CAAGAGGAAAACACAACGTATGATTTGGAAAACGCG
                   AATAAGAAAGCAATTGTTCTGAACTTAAAATCGGAA
                   AAAGGAAAGAAAATTCTTCACGAGATGCTTGCTGAG
                   GCAGACATCTTGTTAACAAATTGGCGCACGAAAGCG
                   TTAGTCAAACAGGGGTTAGATTACGAAACACTGAAA
                   GAGAAGTATCCAAAATTGGTATTTGCACAGATTACAG
                   GATACGGGGAGAAAGGACCCGACAAAGACCTGCCTG
                   GTTTCGACTACACGGCGTTTTTCGCCCGCGGAGGAGT
                   CTCCGGTACATTATATGAAAAAGGAACTGTCCCTCCT
                   AATGTGGTACCGGGTCTGGGTGACCACCAGGCAGGA
                   ATGTTCTTAGCTGCCGGTATGGCTGGTGCGTTGTATA
                   AGGCCAAAACCACCGGACAAGGCGACAAAGTCACCG
                   TTAGTCTGATGCATAGCGCAATGTACGGCCTGGGAAT
                   CATGATTCAGGCAGCCCAGTACAAGGACCATGGGCT
                   GGTGTACCCGATCAACCGTAATGAAACGCCTAATCCT
                   TTCATCGTTTCATACAAGTCCAAAGATGATTACTTTG
                   TCCAAGTTTGCATGCCTCCCTATGATGTGTTTTATGAT
                   CGCTTTATGACGGCCTTAGGACGTGAAGACTTGGTAG
                   GTGACGAACGCTACAATAAGATCGAGAACTTGAAGG
                   ATGGTCGCGCAAAAGAAGTCTATTCCATCATCGAACA
                   ACAAATGGTAACGAAGACGAAGGACGAATGGGACA
                   AGATTTTTCGTGATGCAGACATTCCATTCGCTATTGC
                   CCAAACGTGGGAAGATCTTTTAGAAGACGAGCAGGC
                   ATGGGCCAACGACTACCTGTATAAAATGAAGTATCCC
                   ACAGGCAACGAACGTGCCCTGGTACGTTTACCTGTGT
                   TCTTCAAAGAAGCTGGACTTCCTGAATACAACCAGTC
                   GCCACAGATTGCTGAGAATACCGTGGAAGTGTTAAA
                   GGAGATGGGATATACCGAGCAAGAAATTGAGGAGCT
                   TGAGAAAGACAAAGACATCATGGTACGTAAAGAGAA
                   ATGAAGGTtaagaaggagatatacatATGTCAGACCGCAACAA
                   AGAAGTGAAAGAAAAGAAGGCTAAACACTATCTGCG
                   CGAGATCACAGCTAAACACTACAAGGAAGCGTTAGA
                   GGCTAAAGAGCGTGGGGAGAAAGTGGGTTGGTGTGC
                   CTCTAACTTCCCCCAAGAGATTGCAACCACGTTGGGT
                   GTAAAGGTTGTTTATCCCGAAAACCACGCCGCCGCCG
                   TAGCGGCACGTGGCAATGGGCAAAATATGTGCGAAC
                   ACGCGGAGGCTATGGGATTCAGTAATGATGTGTGTG
                   GATATGCACGTGTAAATTTAGCCGTAATGGACATCGG
                   CCATAGTGAAGATCAACCTATTCCAATGCCTGATTTC
                   GTTCTGTGCTGTAATAATATCTGCAATCAGATGATTA
                   AATGGTATGAACACATTGCAAAAACGTTGGATATTCC
                   TATGATCCTTATCGATATTCCATATAATACTGAGAAC
                   ACGGTGTCTCAGGACCGCATTAAGTACATCCGCGCCC
                   AGTTCGATGACGCTATCAAGCAACTGGAAGAAATCA
                   CTGGCAAAAAGTGGGACGAGAATAAATTCGAAGAAG
                   TGATGAAGATTTCGCAAGAATCGGCCAAGCAATGGT
                   TACGCGCCGCGAGCTACGCGAAATACAAACCATCAC
                   CGTTTTCGGGCTTTGACCTTTTTAATCACATGGCTGTA
                   GCCGTTTGTGCTCGCGGCACCCAGGAAGCCGCCGATG
                   CATTCAAAATGTTAGCAGATGAATATGAAGAGAACG
                   TTAAGACAGGAAAGTCTACTTATCGCGGCGAGGAGA
                   AGCAGCGTATCTTGTTCGAGGGCATCGCTTGTTGGCC
                   TTATCTGCGCCACAAGTTGACGAAACTGAGTGAATAT
                   GGAATGAACGTCACAGCTACGGTGTACGCCGAAGCT
                   TTTGGGGTTATTTACGAAAACATGGATGAACTGATGG
                   CCGCTTACAATAAAGTGCCTAACTCAATCTCCTTCGA
                   GAACGCGCTGAAGATGCGTCTTAATGCCGTTACAAGC
                   ACCAATACAGAAGGGGCTGTTATCCACATTAATCGCA
                   GTTGTAAGCTGTGGTCAGGATTCTTATACGAACTGGC
                   CCGTCGTTTGGAAAAGGAGACGGGGATCCCTGTTGTT
                   TCGTTCGACGGAGATCAAGCGGATCCCCGTAACTTCT
                   CCGAGGCTCAATATGACACTCGCATCCAAGGTTTAAA
                   TGAGGTGATGGTCGCGAAAAAAGAAGCAGAGTGAGC
                   TTtaagaaggagatatacatATGTCGAATAGTGACAAGTTTTTT
                   AACGACTTCAAGGACATTGTGGAAAACCCAAAGAAG
                   TATATCATGAAGCATATGGAACAAACGGGACAAAAA
                   GCCATCGGTTGCATGCCTTTATACACCCCAGAAGAGC
                   TTGTCTTAGCGGCGGGTATGTTTCCTGTTGGAGTATG
                   GGGCTCGAATACTGAGTTGTCAAAAGCCAAGACCTA
                   CTTTCCGGCTTTTATCTGTTCTATCTTGCAAACTACTT
                   TAGAAAACGCATTGAATGGGGAGTATGACATGCTGT
                   CTGGTATGATGATCACAAACTATTGCGATTCGCTGAA
                   ATGTATGGGACAAAACTTCAAACTTACAGTGGAAAA
                   TATCGAATTCATCCCGGTTACGGTTCCACAAAACCGC
                   AAGATGGAGGCGGGTAAAGAATTTCTGAAATCCCAG
                   TATAAAATGAATATCGAACAACTGGAAAAAATCTCA
                   GGGAATAAGATCACTGACGAGAGCTTGGAGAAGGCT
                   ATTGAAATTTACGATGAGCACCGTAAAGTCATGAAC
                   GATTTCTCTATGCTTGCGTCCAAGTACCCTGGTATCAT
                   TACGCCAACGAAACGTAACTACGTGATGAAGTCAGC
                   GTATTATATGGACAAGAAAGAACATACAGAGAAGGT
                   ACGTCAGTTGATGGATGAAATCAAGGCCATTGAGCCT
                   AAACCATTCGAAGGAAAACGCGTGATTACCACTGGG
                   ATCATTGCAGATTCGGAGGACCTTTTGAAAATCTTGG
                   AGGAGAATAACATTGCTATCGTGGGAGATGATATTG
                   CACACGAGTCTCGCCAATACCGCACTTTGACCCCGGA
                   GGCCAACACACCTATGGACCGTCTTGCTGAACAATTT
                   GCGAACCGCGAGTGTTCGACGTTGTATGACCCTGAAA
                   AAAAACGTGGACAGTATATTGTCGAGATGGCAAAAG
                   AGCGTAAGGCCGACGGAATCATCTTCTTCATGACAAA
                   ATTCTGCGATCCCGAAGAATACGATTACCCTCAGATG
                   AAAAAAGACTTCGAAGAAGCCGGTATTCCCCACGTT
                   CTGATTGAGACAGACATGCAAATGAAGAACTACGAA
                   CAAGCTCGCACCGCTATTCAAGCATTTTCAGAAACCC
                   TTTGACGCTtaagaaggagatatacatATGCGTGCTGTCTTAAT
                   CGAGAAGTCAGATGACACCCAGAGTGTTTCAGTTAC
                   GGAGTTGGCTGAAGACCAATTACCCGAAGGTGACGT
                   CCTTGTGGATGTCGCGTACAGCACATTGAATTACAAG
                   GATGCTCTTGCGATTACTGGAAAAGCACCCGTTGTAC
                   GCCGTTTTCCTATGGTCCCCGGAATTGACTTTACTGG
                   GACTGTCGCACAGAGTTCCCATGCTGATTTCAAGCCA
                   GGCGACCGCGTAATTCTGAACGGATGGGGAGTTGGT
                   GAGAAACACTGGGGCGGTCTTGCAGAACGCGCACGC
                   GTACGTGGGGACTGGCTTGTCCCGTTGCCAGCCCCCT
                   TAGACTTGCGCCAGGCTGCAATGATTGGCACTGCGGG
                   GTACACAGCTATGCTGTGCGTGCTTGCCCTTGAGCGC
                   CATGGAGTCGTACCTGGGAACGGCGAGATTGTCGTCT
                   CAGGCGCAGCAGGAGGGGTAGGTTCTGTAGCAACCA
                   CACTGTTAGCAGCCAAAGGCTACGAAGTGGCCGCCG
                   TGACCGGGCGCGCAAGCGAGGCCGAATATTTACGCG
                   GATTAGGCGCCGCGTCGGTCATTGATCGCAATGAATT
                   AACGGGGAAGGTGCGTCCATTAGGGCAGGAACGCTG
                   GGCAGGAGGAATCGATGTAGCAGGATCAACCGTACT
                   TGCTAATATGTTGAGCATGATGAAATACCGTGGCGTG
                   GTGGCGGCCTGTGGCCTGGCGGCTGGAATGGACTTGC
                   CCGCGTCTGTCGCCCCTTTTATTCTGCGTGGTATGACT
                   TTGGCAGGGGTAGATTCAGTCATGTGCCCCAAAACTG
                   ATCGTCTGGCTGCTTGGGCACGCCTGGCATCCGACCT
                   GGACCCTGCAAAGCTGGAAGAGATGACAACTGAATT
                   ACCGTTCTCTGAGGTGATTGAAACGGCTCCGAAGTTC
                   TTGGATGGAACAGTGCGTGGGCGTATTGTCATTCCGG
                   TAACACCTTGATACTtaagaaggagatatacatATGAAAATCTT
                   GGCATACTGCGTCCGCCCAGACGAGGTAGACTCCTTT
                   AAGAAATTTAGTGAAAAGTACGGGCATACAGTTGAT
                   CTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATTT
                   GGCGAAGGGTTACGATGGGATTTCTATTCTGGGCAAC
                   GACACGTGTAACCGTGAGGCACTGGAGAAGATCAAG
                   GATTGCGGGATCAAATATCTGGCAACCCGTACAGCC
                   GGAGTGAACAACATTGACTTCGATGCAGCAAAGGAG
                   TTCGGTATTAACGTGGCTAATGTTCCCGCATATTCCC
                   CCAACTCGGTCAGCGAATTTACCATTGGATTGGCATT
                   AAGTCTGACGCGTAAGATTCCATTTGCCCTGAAACGC
                   GTGGAACTGAACAATTTTGCGCTTGGCGGCCTTATTG
                   GTGTGGAATTGCGTAACTTAACTTTAGGAGTCATCGG
                   TACTGGTCGCATCGGATTGAAAGTGATTGAGGGCTTC
                   TCTGGGTTTGGAATGAAAAAAATGATCGGTTATGACA
                   TTTTTGAAAATGAAGAAGCAAAGAAGTACATCGAAT
                   ACAAATCATTAGACGAAGTTTTTAAAGAGGCTGATAT
                   TATCACTCTGCATGCGCCTCTGACAGACGACAACTAT
                   CATATGATTGGTAAAGAATCCATTGCTAAAATGAAG
                   GATGGGGTATTTATTATCAACGCAGCGCGTGGAGCCT
                   TAATCGATAGTGAGGCCCTGATTGAAGGGTTAAAATC
                   GGGGAAGATT
fldA               ATGGAAAACAACACCAATATGTTCTCTGGAGTGAAG
SEQ ID NO: 276     GTGATCGAACTGGCCAACTTTATCGCTGCTCCGGCGG
                   CAGGTCGCTTCTTTGCTGATGGGGGAGCAGAAGTAAT
                   TAAGATCGAATCTCCAGCAGGCGACCCGCTGCGCTAC
                   ACGGCCCCATCAGAAGGACGCCCGCTTTCTCAAGAG
                   GAAAACACAACGTATGATTTGGAAAACGCGAATAAG
                   AAAGCAATTGTTCTGAACTTAAAATCGGAAAAAGGA
                   AAGAAAATTCTTCACGAGATGCTTGCTGAGGCAGAC
                   ATCTTGTTAACAAATTGGCGCACGAAAGCGTTAGTCA
                   AACAGGGGTTAGATTACGAAACACTGAAAGAGAAGT
                   ATCCAAAATTGGTATTTGCACAGATTACAGGATACGG
                   GGAGAAAGGACCCGACAAAGACCTGCCTGGTTTCGA
                   CTACACGGCGTTTTTCGCCCGCGGAGGAGTCTCCGGT
                   ACATTATATGAAAAAGGAACTGTCCCTCCTAATGTGG
                   TACCGGGTCTGGGTGACCACCAGGCAGGAATGTTCTT
                   AGCTGCCGGTATGGCTGGTGCGTTGTATAAGGCCAAA
                   ACCACCGGACAAGGCGACAAAGTCACCGTTAGTCTG
                   ATGCATAGCGCAATGTACGGCCTGGGAATCATGATTC
                   AGGCAGCCCAGTACAAGGACCATGGGCTGGTGTACC
                   CGATCAACCGTAATGAAACGCCTAATCCTTTCATCGT
                   TTCATACAAGTCCAAAGATGATTACTTTGTCCAAGTT
                   TGCATGCCTCCCTATGATGTGTTTTATGATCGCTTTAT
                   GACGGCCTTAGGACGTGAAGACTTGGTAGGTGACGA
                   ACGCTACAATAAGATCGAGAACTTGAAGGATGGTCG
                   CGCAAAAGAAGTCTATTCCATCATCGAACAACAAAT
                   GGTAACGAAGACGAAGGACGAATGGGACAAGATTTT
                   TCGTGATGCAGACATTCCATTCGCTATTGCCCAAACG
                   TGGGAAGATCTTTTAGAAGACGAGCAGGCATGGGCC
                   AACGACTACCTGTATAAAATGAAGTATCCCACAGGC
                   AACGAACGTGCCCTGGTACGTTTACCTGTGTTCTTCA
                   AAGAAGCTGGACTTCCTGAATACAACCAGTCGCCAC
                   AGATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGA
                   TGGGATATACCGAGCAAGAAATTGAGGAGCTTGAGA
                   AAGACAAAGACATCATGGTACGTAAAGAGAAATGA
fldB               ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA
SEQ ID NO: 277     GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA
                   CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA
                   GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG
                   ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG
                   AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG
                   GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT
                   TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT
                   AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC
                   TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA
                   TCTGCAATCAGATGATTAAATGGTATGAACACATTGC
                   AAAAACGTTGGATATTCCTATGATCCTTATCGATATT
                   CCATATAATACTGAGAACACGGTGTCTCAGGACCGC
                   ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA
                   AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG
                   AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG
                   AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG
                   CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT
                   TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC
                   ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA
                   GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT
                   ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG
                   AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT
                   GACGAAACTGAGTGAATATGGAATGAACGTCACAGC
                   TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA
                   AACATGGATGAACTGATGGCCGCTTACAATAAAGTG
                   CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC
                   GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG
                   CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC
                   AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG
                   GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC
                   AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA
                   CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG
                   AAAAAAGAAGCAGAGTGA
fldC               ATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAGG
SEQ ID NO: 278     ACATTGTGGAAAACCCAAAGAAGTATATCATGAAGC
                   ATATGGAACAAACGGGACAAAAAGCCATCGGTTGCA
                   TGCCTTTATACACCCCAGAAGAGCTTGTCTTAGCGGC
                   GGGTATGTTTCCTGTTGGAGTATGGGGCTCGAATACT
                   GAGTTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTA
                   TCTGTTCTATCTTGCAAACTACTTTAGAAAACGCATT
                   GAATGGGGAGTATGACATGCTGTCTGGTATGATGATC
                   ACAAACTATTGCGATTCGCTGAAATGTATGGGACAA
                   AACTTCAAACTTACAGTGGAAAATATCGAATTCATCC
                   CGGTTACGGTTCCACAAAACCGCAAGATGGAGGCGG
                   GTAAAGAATTTCTGAAATCCCAGTATAAAATGAATAT
                   CGAACAACTGGAAAAAATCTCAGGGAATAAGATCAC
                   TGACGAGAGCTTGGAGAAGGCTATTGAAATTTACGA
                   TGAGCACCGTAAAGTCATGAACGATTTCTCTATGCTT
                   GCGTCCAAGTACCCTGGTATCATTACGCCAACGAAAC
                   GTAACTACGTGATGAAGTCAGCGTATTATATGGACAA
                   GAAAGAACATACAGAGAAGGTACGTCAGTTGATGGA
                   TGAAATCAAGGCCATTGAGCCTAAACCATTCGAAGG
                   AAAACGCGTGATTACCACTGGGATCATTGCAGATTCG
                   GAGGACCTTTTGAAAATCTTGGAGGAGAATAACATT
                   GCTATCGTGGGAGATGATATTGCACACGAGTCTCGCC
                   AATACCGCACTTTGACCCCGGAGGCCAACACACCTAT
                   GGACCGTCTTGCTGAACAATTTGCGAACCGCGAGTGT
                   TCGACGTTGTATGACCCTGAAAAAAAACGTGGACAG
                   TATATTGTCGAGATGGCAAAAGAGCGTAAGGCCGAC
                   GGAATCATCTTCTTCATGACAAAATTCTGCGATCCCG
                   AAGAATACGATTACCCTCAGATGAAAAAAGACTTCG
                   AAGAAGCCGGTATTCCCCACGTTCTGATTGAGACAGA
                   CATGCAAATGAAGAACTACGAACAAGCTCGCACCGC
                   TATTCAAGCATTTTCAGAAACCCTTTG
AcuI               ATGCGTGCTGTCTTAATCGAGAAGTCAGATGACACCC
SEQ ID NO: 279     AGAGTGTTTCAGTTACGGAGTTGGCTGAAGACCAATT
                   ACCCGAAGGTGACGTCCTTGTGGATGTCGCGTACAGC
                   ACATTGAATTACAAGGATGCTCTTGCGATTACTGGAA
                   AAGCACCCGTTGTACGCCGTTTTCCTATGGTCCCCGG
                   AATTGACTTTACTGGGACTGTCGCACAGAGTTCCCAT
                   GCTGATTTCAAGCCAGGCGACCGCGTAATTCTGAACG
                   GATGGGGAGTTGGTGAGAAACACTGGGGCGGTCTTG
                   CAGAACGCGCACGCGTACGTGGGGACTGGCTTGTCC
                   CGTTGCCAGCCCCCTTAGACTTGCGCCAGGCTGCAAT
                   GATTGGCACTGCGGGGTACACAGCTATGCTGTGCGTG
                   CTTGCCCTTGAGCGCCATGGAGTCGTACCTGGGAACG
                   GCGAGATTGTCGTCTCAGGCGCAGCAGGAGGGGTAG
                   GTTCTGTAGCAACCACACTGTTAGCAGCCAAAGGCTA
                   CGAAGTGGCCGCCGTGACCGGGCGCGCAAGCGAGGC
                   CGAATATTTACGCGGATTAGGCGCCGCGTCGGTCATT
                   GATCGCAATGAATTAACGGGGAAGGTGCGTCCATTA
                   GGGCAGGAACGCTGGGCAGGAGGAATCGATGTAGCA
                   GGATCAACCGTACTTGCTAATATGTTGAGCATGATGA
                   AATACCGTGGCGTGGTGGCGGCCTGTGGCCTGGCGG
                   CTGGAATGGACTTGCCCGCGTCTGTCGCCCCTTTTATT
                   CTGCGTGGTATGACTTTGGCAGGGGTAGATTCAGTCA
                   TGTGCCCCAAAACTGATCGTCTGGCTGCTTGGGCACG
                   CCTGGCATCCGACCTGGACCCTGCAAAGCTGGAAGA
                   GATGACAACTGAATTACCGTTCTCTGAGGTGATTGAA
                   ACGGCTCCGAAGTTCTTGGATGGAACAGTGCGTGGG
                   CGTATTGTCATTCCGGTAACACCTTGA
fldH1              ATGAAAATCTTGGCATACTGCGTCCGCCCAGACGAG
SEQ ID NO: 280     GTAGACTCCTTTAAGAAATTTAGTGAAAAGTACGGGC
                   ATACAGTTGATCTTATTCCAGACTCTTTTGGACCTAAT
                   GTCGCTCATTTGGCGAAGGGTTACGATGGGATTTCTA
                   TTCTGGGCAACGACACGTGTAACCGTGAGGCACTGG
                   AGAAGATCAAGGATTGCGGGATCAAATATCTGGCAA
                   CCCGTACAGCCGGAGTGAACAACATTGACTTCGATGC
                   AGCAAAGGAGTTCGGTATTAACGTGGCTAATGTTCCC
                   GCATATTCCCCCAACTCGGTCAGCGAATTTACCATTG
                   GATTGGCATTAAGTCTGACGCGTAAGATTCCATTTGC
                   CCTGAAACGCGTGGAACTGAACAATTTTGCGCTTGGC
                   GGCCTTATTGGTGTGGAATTGCGTAACTTAACTTTAG
                   GAGTCATCGGTACTGGTCGCATCGGATTGAAAGTGAT
                   TGAGGGCTTCTCTGGGTTTGGAATGAAAAAAATGATC
                   GGTTATGACATTTTTGAAAATGAAGAAGCAAAGAAG
                   TACATCGAATACAAATCATTAGACGAAGTTTTTAAAG
                   AGGCTGATATTATCACTCTGCATGCGCCTCTGACAGA
                   CGACAACTATCATATGATTGGTAAAGAATCCATTGCT
                   AAAATGAAGGATGGGGTATTTATTATCAACGCAGCG
                   CGTGGAGCCTTAATCGATAGTGAGGCCCTGATTGAAG
                   GGTTAAAATCGGGGAAGATTGCGGGCGCGGCTCTGG
                   ATAGCTATGAGTATGAGCAAGGTGTCTTTCACAACAA
                   TAAGATGAATGAAATTATGCAGGATGATACCTTGGA
                   ACGTCTGAAATCTTTTCCCAACGTCGTGATCACGCCG
                   CATTTGGGTTTTTATACTGATGAGGCGGTTTCCAATA
                   TGGTAGAGATCACACTGATGAACCTTCAGGAATTCGA
                   GTTGAAAGGAACCTGTAAGAACCAGCGTGTTTGTAA
                   ATGA
fbrAroG-TrpDH-     Ctctagaaataattttgtttaactttaagaaggagatatacat
fldABCDH (RBS      atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg
and leader region  cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga
underlined)        aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca
SEQ ID NO: 281     tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg
                   aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta
                   cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac
                   gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg
                   gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc
                   gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc
                   ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta
                   atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt
                   gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa
                   ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc
                   agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat
                   ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt
                   gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct
                   ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt
                   acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaaggaga
                   tatacatATGCTGTTATTCGAGACTGTGCGTGAAATGGGT
                   CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG
                   AGATCAAGGCAATTATCGCAATCCACGATACCACCTT
                   AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT
                   ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC
                   TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA
                   ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC
                   TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC
                   ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC
                   ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC
                   GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG
                   GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA
                   CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT
                   AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT
                   GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA
                   AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA
                   CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG
                   AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC
                   GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC
                   CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT
                   CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG
                   AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG
                   ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT
                   ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA
                   CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA
                   ACAAGTTCATAACATCTACGATACGTTATTAGCGATT
                   TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC
                   GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC
                   AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG
                   TGAAATGtaagaaggagatatacatATGGAAAACAACACCAAT
                   ATGTTCTCTGGAGTGAAGGTGATCGAACTGGCCAACT
                   TTATCGCTGCTCCGGCGGCAGGTCGCTTCTTTGCTGA
                   TGGGGGAGCAGAAGTAATTAAGATCGAATCTCCAGC
                   AGGCGACCCGCTGCGCTACACGGCCCCATCAGAAGG
                   ACGCCCGCTTTCTCAAGAGGAAAACACAACGTATGA
                   TTTGGAAAACGCGAATAAGAAAGCAATTGTTCTGAA
                   CTTAAAATCGGAAAAAGGAAAGAAAATTCTTCACGA
                   GATGCTTGCTGAGGCAGACATCTTGTTAACAAATTGG
                   CGCACGAAAGCGTTAGTCAAACAGGGGTTAGATTAC
                   GAAACACTGAAAGAGAAGTATCCAAAATTGGTATTT
                   GCACAGATTACAGGATACGGGGAGAAAGGACCCGAC
                   AAAGACCTGCCTGGTTTCGACTACACGGCGTTTTTCG
                   CCCGCGGAGGAGTCTCCGGTACATTATATGAAAAAG
                   GAACTGTCCCTCCTAATGTGGTACCGGGTCTGGGTGA
                   CCACCAGGCAGGAATGTTCTTAGCTGCCGGTATGGCT
                   GGTGCGTTGTATAAGGCCAAAACCACCGGACAAGGC
                   GACAAAGTCACCGTTAGTCTGATGCATAGCGCAATGT
                   ACGGCCTGGGAATCATGATTCAGGCAGCCCAGTACA
                   AGGACCATGGGCTGGTGTACCCGATCAACCGTAATG
                   AAACGCCTAATCCTTTCATCGTTTCATACAAGTCCAA
                   AGATGATTACTTTGTCCAAGTTTGCATGCCTCCCTAT
                   GATGTGTTTTATGATCGCTTTATGACGGCCTTAGGAC
                   GTGAAGACTTGGTAGGTGACGAACGCTACAATAAGA
                   TCGAGAACTTGAAGGATGGTCGCGCAAAAGAAGTCT
                   ATTCCATCATCGAACAACAAATGGTAACGAAGACGA
                   AGGACGAATGGGACAAGATTTTTCGTGATGCAGACA
                   TTCCATTCGCTATTGCCCAAACGTGGGAAGATCTTTT
                   AGAAGACGAGCAGGCATGGGCCAACGACTACCTGTA
                   TAAAATGAAGTATCCCACAGGCAACGAACGTGCCCT
                   GGTACGTTTACCTGTGTTCTTCAAAGAAGCTGGACTT
                   CCTGAATACAACCAGTCGCCACAGATTGCTGAGAAT
                   ACCGTGGAAGTGTTAAAGGAGATGGGATATACCGAG
                   CAAGAAATTGAGGAGCTTGAGAAAGACAAAGACATC
                   ATGGTACGTAAAGAGAAATGAAGGTtaagaaggagatatacat
                   ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA
                   GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA
                   CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA
                   GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG
                   ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG
                   AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG
                   GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT
                   TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT
                   AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC
                   TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA
                   TCTGCAATCAGATGATTAAATGGTATGAACACATTGC
                   AAAAACGTTGGATATTCCTATGATCCTTATCGATATT
                   CCATATAATACTGAGAACACGGTGTCTCAGGACCGC
                   ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA
                   AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG
                   AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG
                   AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG
                   CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT
                   TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC
                   ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA
                   GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT
                   ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG
                   AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT
                   GACGAAACTGAGTGAATATGGAATGAACGTCACAGC
                   TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA
                   AACATGGATGAACTGATGGCCGCTTACAATAAAGTG
                   CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC
                   GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG
                   CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC
                   AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG
                   GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC
                   AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA
                   CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG
                   AAAAAAGAAGCAGAGTGAGCTTtaagaaggagatatacatATG
                   TCGAATAGTGACAAGTTTTTTAACGACTTCAAGGACA
                   TTGTGGAAAACCCAAAGAAGTATATCATGAAGCATA
                   TGGAACAAACGGGACAAAAAGCCATCGGTTGCATGC
                   CTTTATACACCCCAGAAGAGCTTGTCTTAGCGGCGGG
                   TATGTTTCCTGTTGGAGTATGGGGCTCGAATACTGAG
                   TTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTATCT
                   GTTCTATCTTGCAAACTACTTTAGAAAACGCATTGAA
                   TGGGGAGTATGACATGCTGTCTGGTATGATGATCACA
                   AACTATTGCGATTCGCTGAAATGTATGGGACAAAACT
                   TCAAACTTACAGTGGAAAATATCGAATTCATCCCGGT
                   TACGGTTCCACAAAACCGCAAGATGGAGGCGGGTAA
                   AGAATTTCTGAAATCCCAGTATAAAATGAATATCGAA
                   CAACTGGAAAAAATCTCAGGGAATAAGATCACTGAC
                   GAGAGCTTGGAGAAGGCTATTGAAATTTACGATGAG
                   CACCGTAAAGTCATGAACGATTTCTCTATGCTTGCGT
                   CCAAGTACCCTGGTATCATTACGCCAACGAAACGTAA
                   CTACGTGATGAAGTCAGCGTATTATATGGACAAGAA
                   AGAACATACAGAGAAGGTACGTCAGTTGATGGATGA
                   AATCAAGGCCATTGAGCCTAAACCATTCGAAGGAAA
                   ACGCGTGATTACCACTGGGATCATTGCAGATTCGGAG
                   GACCTTTTGAAAATCTTGGAGGAGAATAACATTGCTA
                   TCGTGGGAGATGATATTGCACACGAGTCTCGCCAATA
                   CCGCACTTTGACCCCGGAGGCCAACACACCTATGGAC
                   CGTCTTGCTGAACAATTTGCGAACCGCGAGTGTTCGA
                   CGTTGTATGACCCTGAAAAAAAACGTGGACAGTATA
                   TTGTCGAGATGGCAAAAGAGCGTAAGGCCGACGGAA
                   TCATCTTCTTCATGACAAAATTCTGCGATCCCGAAGA
                   ATACGATTACCCTCAGATGAAAAAAGACTTCGAAGA
                   AGCCGGTATTCCCCACGTTCTGATTGAGACAGACATG
                   CAAATGAAGAACTACGAACAAGCTCGCACCGCTATT
                   CAAGCATTTTCAGAAACCCTTTGACGCTtaagaaggagatata
                   catATGTTCTTTACGGAGCAACACGAACTTATTCGCAA
                   ACTGGCGCGTGACTTTGCCGAACAGGAAATCGAGCC
                   TATCGCAGACGAAGTAGATAAAACCGCAGAGTTCCC
                   AAAAGAAATCGTGAAGAAGATGGCTCAAAATGGATT
                   TTTCGGCATTAAAATGCCTAAAGAATACGGAGGGGC
                   GGGTGCGGATAACCGCGCTTATGTCACTATTATGGAG
                   GAAATTTCACGTGCTTCCGGGGTAGCGGGTATCTACC
                   TGAGCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTT
                   ATTGGTCGGAACCGATGAGCAAAAAGAAAAGTACCT
                   TAAGCCTATGATCCGCGGCGAGAAGACTCTGGCGTTC
                   GCCCTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTG
                   CGTTGGCTACTACTGCCCGTGAAGAGGGCGACTATTA
                   TATCTTAAATGGCCGCAAGACGTTTATTACAGGGGCT
                   CCTATTAGCGACAATATTATTGTGTTCGCAAAAACCG
                   ATATGAGCAAAGGGACCAAAGGTATCACCACTTTCA
                   TTGTGGACTCAAAGCAGGAAGGGGTAAGTTTTGGTA
                   AGCCAGAGGACAAAATGGGAATGATTGGTTGTCCGA
                   CAAGCGACATCATCTTGGAAAACGTTAAAGTTCATAA
                   GTCCGACATCTTGGGAGAAGTCAATAAGGGGTTTATT
                   ACCGCGATGAAAACACTTTCCGTTGGTCGTATCGGAG
                   TGGCGTCACAGGCGCTTGGAATTGCACAGGCCGCCGT
                   AGATGAGGCGGTAAAGTACGCCAAGCAACGTAAACA
                   ATTCAATCGCCCAATCGCGAAATTTCAGGCCATTCAA
                   TTTAAACTTGCCAATATGGAGACTAAATTAAATGCCG
                   CTAAACTTCTTGTTTATAACGCAGCGTACAAAATGGA
                   TTGTGGAGAAAAAGCCGACAAGGAAGCCTCTATGGC
                   TAAATACTTTGCTGCTGAATCAGCGATCCAAATCGTT
                   AACGACGCGCTGCAAATCCATGGCGGGTATGGCTAT
                   ATCAAAGACTACAAGATTGAACGTTTGTACCGCGATG
                   TGCGTGTGATCGCTATTTATGAGGGCACTTCCGAGGT
                   CCAACAGATGGTTATCGCGTCCAATCTGCTGAAGTAA
                   TACTtaagaaggagatatacatATGAAAATCTTGGCATACTGCG
                   TCCGCCCAGACGAGGTAGACTCCTTTAAGAAATTTAG
                   TGAAAAGTACGGGCATACAGTTGATCTTATTCCAGAC
                   TCTTTTGGACCTAATGTCGCTCATTTGGCGAAGGGTT
                   ACGATGGGATTTCTATTCTGGGCAACGACACGTGTAA
                   CCGTGAGGCACTGGAGAAGATCAAGGATTGCGGGAT
                   CAAATATCTGGCAACCCGTACAGCCGGAGTGAACAA
                   CATTGACTTCGATGCAGCAAAGGAGTTCGGTATTAAC
                   GTGGCTAATGTTCCCGCATATTCCCCCAACTCGGTCA
                   GCGAATTTACCATTGGATTGGCATTAAGTCTGACGCG
                   TAAGATTCCATTTGCCCTGAAACGCGTGGAACTGAAC
                   AATTTTGCGCTTGGCGGCCTTATTGGTGTGGAATTGC
                   GTAACTTAACTTTAGGAGTCATCGGTACTGGTCGCAT
                   CGGATTGAAAGTGATTGAGGGCTTCTCTGGGTTTGGA
                   ATGAAAAAAATGATCGGTTATGACATTTTTGAAAATG
                   AAGAAGCAAAGAAGTACATCGAATACAAATCATTAG
                   ACGAAGTTTTTAAAGAGGCTGATATTATCACTCTGCA
                   TGCGCCTCTGACAGACGACAACTATCATATGATTGGT
                   AAAGAATCCATTGCTAAAATGAAGGATGGGGTATTT
                   ATTATCAACGCAGCGCGTGGAGCCTTAATCGATAGTG
                   AGGCCCTGATTGAAGGGTTAAAATCGGGGAAGATTG
                   CGGGCGCGGCTCTGGATAGCTATGAGTATGAGCAAG
                   GTGTCTTTCACAACAATAAGATGAATGAAATTATGCA
                   GGATGATACCTTGGAACGTCTGAAATCTTTTCCCAAC
                   GTCGTGATCACGCCGCATTTGGGTTTTTATACTGATG
                   AGGCGGTTTCCAATATGGTAGAGATCACACTGATGA
                   ACCTTCAGGAATTCGAGTTGAAAGGAACCTGTAAGA
                   ACCAGCGTGTTTGTAAATGA
FldD               ATGTTCTTTACGGAGCAACACGAACTTATTCGCAAAC
SEQ ID NO: 282     TGGCGCGTGACTTTGCCGAACAGGAAATCGAGCCTAT
                   CGCAGACGAAGTAGATAAAACCGCAGAGTTCCCAAA
                   AGAAATCGTGAAGAAGATGGCTCAAAATGGATTTTT
                   CGGCATTAAAATGCCTAAAGAATACGGAGGGGCGGG
                   TGCGGATAACCGCGCTTATGTCACTATTATGGAGGAA
                   ATTTCACGTGCTTCCGGGGTAGCGGGTATCTACCTGA
                   GCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTTATT
                   GGTCGGAACCGATGAGCAAAAAGAAAAGTACCTTAA
                   GCCTATGATCCGCGGCGAGAAGACTCTGGCGTTCGCC
                   CTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTGCGT
                   TGGCTACTACTGCCCGTGAAGAGGGCGACTATTATAT
                   CTTAAATGGCCGCAAGACGTTTATTACAGGGGCTCCT
                   ATTAGCGACAATATTATTGTGTTCGCAAAAACCGATA
                   TGAGCAAAGGGACCAAAGGTATCACCACTTTCATTGT
                   GGACTCAAAGCAGGAAGGGGTAAGTTTTGGTAAGCC
                   AGAGGACAAAATGGGAATGATTGGTTGTCCGACAAG
                   CGACATCATCTTGGAAAACGTTAAAGTTCATAAGTCC
                   GACATCTTGGGAGAAGTCAATAAGGGGTTTATTACCG
                   CGATGAAAACACTTTCCGTTGGTCGTATCGGAGTGGC
                   GTCACAGGCGCTTGGAATTGCACAGGCCGCCGTAGA
                   TGAGGCGGTAAAGTACGCCAAGCAACGTAAACAATT
                   CAATCGCCCAATCGCGAAATTTCAGGCCATTCAATTT
                   AAACTTGCCAATATGGAGACTAAATTAAATGCCGCTA
                   AACTTCTTGTTTATAACGCAGCGTACAAAATGGATTG
                   TGGAGAAAAAGCCGACAAGGAAGCCTCTATGGCTAA
                   ATACTTTGCTGCTGAATCAGCGATCCAAATCGTTAAC
                   GACGCGCTGCAAATCCATGGCGGGTATGGCTATATCA
                   AAGACTACAAGATTGAACGTTTGTACCGCGATGTGCG
                   TGTGATCGCTATTTATGAGGGCACTTCCGAGGTCCAA
                   CAGATGGTTATCGCGTCCAATCTGCTGAAGTAA
RBS                taagaaggagatatacat
SEQ ID NO: 283
RBS                ctctagaaataattttgtttaactttaagaaggagatatacat
SEQ ID NO: 284

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 59. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 59. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 59. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 59. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 59. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 59. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 59.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 263. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 263.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 261. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 261. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 261.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 273. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 273. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 273.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 256. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 256. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 256.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 257. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 257. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 257.

Example 38

Tryptophan Production in an Engineered Strain of E. coli Nissle

A number of tryptophan metabolites, either host-derived (such as tryptamine or kynurenine) or intestinal bacteria-derived (such as indole acetate or indole), have been shown to downregulate inflammation and promote gut barrier health, via the activation of the AhR receptor. Other tryptophan metabolites, such as the bacteria-derived indole propionate, have been shown to help restore intestinal barrier integrity, in experimental models of colitis. In this example, the E. coli strain Nissle was engineered to produce tryptophan, the precursor to all those beneficial metabolites.

First, in order to remove the negative regulation of tryptophan biosynthetic genes mediated by the transcription factor TrpR, the trpR gene was deleted form the E. coli Nissle genome. The tryptophan operon trpEDCBA was amplified by PCR from the E. coli Nissle genomic DNA and cloned in the low-copy plasmid pSC101 under the control of the tet promoter, downstream of the tetR repressor gene. This tet-trpEDCBA plasmid was then transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpEDCBA strain. Subsequently, a feedback resistant version of the aroG gene (aroG^fbr) from E. coli Nissle, coding for the enzyme catalyzing the first committing step towards aromatic amino acid production, was synthetized and cloned into the medium copy plasmid p15A, under the control of the tet promoter, downstream of the tetR repressor. This plasmid was transformed into the ΔtrpR, tet-trpEDCBA strain to obtain the ΔtrpR, tet-trpEDCBA, tet-aroG^fbr strain. Finally, a feedback resistant version of the tet-trpEBCDA construct (tet-trpE^fbrBCDA) was generated from the tet-trpEBCDA. Both the tet-aroG^fbr and the tet-trpE^fbrBCDA constructs were transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpE^fbrDCBA, tet-aroG^fbr strain.

All generated strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 0.5% glucose and placed at 37 C at 250 rpm. 200 uL were collected at 2 h, 4 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41A shows that tryptophan is being produced and secreted by the ΔtrpR, tet-trpEDCBA, tet-aroG^fbr strain. The production of tryptophan is significantly enhanced by expressing the feedback resistant version of trpE.

Example 39

Improved Tryptophan by Using a non-PTS Carbon Source and by Deleting the tnaA Gene Encoding Tryptophanase

One of the precursor molecule to tryptophan in E. coli is phosphoenolpyruvate (PEP). Only 3% of available PEP is normally used to produce aromatic acids (that include tryptophan, phenylalanine and tyrosine). When E. coli is grown using glucose as a sole carbon source, 50% of PEP is used to import glucose into the cell using the phosphotransferase system (PTS). In order to increase tryptophan production, a non-PTS oxidized sugar, glucuronate, was used to test tryptophan secretion by the engineered E. coli Nissle strain ΔrpR, tet-trpe^fbrDCBA, tet-aroG^fbr. In addition, the tnaA gene, encoding the tryptophanase enzyme, was deleted in the ΔtrpR, tet-trpE^fbrDCBA, tet-aroG^fbr strain in order to block the conversion of tryptophan into indole to obtain the ΔtrpRΔtnaA, tet-trpE ^fbrDCBA, tet-aroG^fbr strain.

the ΔtrpR, tet-trpE^fbrDCBA, tet-aroG^fbr and ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-arod^br strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucose or 1% glucuronate and placed at 37 C at 250 rpm or at 37 C in an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41B shows that tryptophan production is doubled in aerobic condition when the non-PTS oxidized sugar glucoronate was used. In addition, the deletion of tnaA had a positive effect on tryptophan production at the 3 h time point in both aerobic and anaerobic conditions and at the 16 h time point, only in anaerobic condition.

Example 40

Improved Tryptophan Production by Increasing the Rate of Serine Biosynthesis in E. coli Nissle

The last step in the tryptophan biosynthesis in E. coli consumes one molecule of serine. In this example, we demonstrate that serine availability is a limiting factor for tryptophan production and describe the construction of the tryptophan producing E. coli Nissle strains ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-aroG^fbrserA and ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-aroG^fbrserA^fbr strains.

the ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-aroG^fbr strain was grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucuronate or 1% glucuronate and 10 mM serine and placed at 37 C an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41C shows that tryptophan production is improved three-fold by serine addition.

In order to increase the rate of serine biosynthesis in the ΔtrpRΔtnaA, tet-trpE ^fbrDCBA, tet-aroG^fbr strain, the serA gene from E. coli Nissle encoding the enzyme catalyzing the first step in the serine biosynthetic pathway was amplified by PCR and cloned into the tet-aroG^fbr plasmid by Gibson assembly. The newly generated tet-aroG^fbr-serA construct was then transformed into a ΔtrpRΔtnaA, tet-trpE^fbrDCBA strain to generate the ΔtrpRΔtnaA, tet-trpE^fbrDCBA, tet-aroG^fbr-serA strain. The tet-aroG^fbr-serA construct was further modified to encode a feedback resistant version of serA (serA^fbr). The newly generated tet-aroG^fbr-serA^fbr construct was used to produce the ΔtrpRΔtnaA, tet-trpE ^fbrDCBA, tet-aroG^fbr-serA^fbr strain, optimized to improve the rate of serine biosynthesis and maximize tryptophan production.

Example 41

Comparison of Various Tryptophan Producing Strains

Compare the rates of tryptophan production in the different strains generated, the following constructs and strains were generated according to methods and sequences described herein (e.g. Example 42), and assayed for tryptophan production in the presence of glucuronate as a carbon source under aerobic conditions. SYN2126 comprises ΔtrpRΔtnaA (ΔtrpRΔtnaA). SYN2323 comprises ΔtrpRΔtnaA and a tetracycline inducible construct for the expression of feedback resistant aroG on a plasmid (AtrpRΔtnaA, tet-aroGfbr). SYN2339 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEfbrDCBA). SYN2473 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG and SerA on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA). SYN2476 comprises ΔtrpRΔtnaA and a tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a plasmid (AtrpRΔtnaA, tet-trpEfbrDCBA).

Overnight cultures were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Next, cells were induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm), spun down, washed with cmL M9, spun down again and resuspended in 3 mL M9+1% glucuronate. Cells were plated for CFU counting. For the assay, the cells were placed of 37 C with shaking at 250 rpm. Supernatants were collected at 1 h, 2 h, 3 h, 4 h 16 h for HPLC analysis for tryptophan. As seen in FIG. 42, results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

Example 42

Bacterial Production of Indole Acetic Acid (IAA)

The ability of a strain comprising tryptophan production circuits and additionally Indole-3-pyruvate decarboxylase from Enterobacter cloacae (IpdC) and Indole-3-acetaldehyde dehydrogenase from Ustilago maydis (Iadl) to produce indole acetic acid (IAA) was tested. The following strains were generated according to methods described herein and tested.

SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan; ΔtrpR and ΔtnaA, a first tetracline inducible trpEfbrDCBA construct on a first plasmid(pSC101), and a second tetracycline inducible aroGfbr construct on a second plasmid (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)) (FIG. 36B). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises trpDH-ipdC-iadl incorporated at the end of the second construct (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A))(FIG. 39B).

Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 1 h, 2.5 h and 18 h for LCMS analysis of tryptophan and indole acetic acid as described herein.

As seen in FIG. 45, SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 containing the additional IAA producing circuitry produces amounts of IAA that are comparable to the amounts of tryptophan produced in its parent SYN2339. No tryptophan is measured, indicating that all tryptophan produced in SYN2342 is efficiently converted into IAA.

Example 43

Tryptamine Production Comparing Two Tryptophan Decarboxylases

The efficacy of two tryptophan decarboxylases (tdc), one from Catharanthus roseus (tdc_Cr)and a second from Clostridium sporogenes (tdc_Cs) in producing tryptamine from tryptophan was tested. The following strains were generated according to methods described herein and tested.

SYN2339 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^thrDCBA construct on a plasmid and another tetracycline inducible construct expressing aroG^fbr on a second plasmid (ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tetaroG^fbr (p15A)). SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine. SYN2340 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^thrDCBA construct on a plasmid and another tetracycline inducible construct expressing aroG^fbr tdc_Cr on a second plasmid (ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tetaroG^fbr-tdc_Cr (p15A)). SYN2794 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^thrDCBA construct on a plasmid and another tetracycline inducible construct expressing arod^k tdc_Cs on a second plasmid (ΔtrpRΔtnaA, tetR-P_tet-trpE^fbrDCBA (pSC101), tetR-P_tet-aroG^fbr-tdc_Cs (p15A)).

Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 3 h and 18 h for LCMS analysis of tryptophan and tryptamine, as described herein.

As seen in FIG. 46, Tdc_Cs from Clostridium sporogenes is more efficient than Tdc_Cr from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine

Example 44

Tryptophan and Anthranilic Acid Quantification in Bacterial Supernatant by LC-MS/MS

Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032m/mL) were prepared in water. Samples (10 μL) and standards were mixed with 90 μL of ACN/H20 (60:30, v/v) containing 1 μg/mL of Tryptophan-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.

LC-MS/MS Method

Tryptophan and Anthranilic acid were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 60., Table 61, and Table 62 provide the summary of the LC-MS/MS method.

TABLE 60
HPLC Method
Column           Accucore aQ column, 2.6 μm
                 (100 × 2.1 mm)
Mobile Phase A   99.9% H2O, 0.1% Formic Acid
Mobile Phase B   99.9% ACN, 0.1% Formic Acid
Injection volume 10 uL

TABLE 61
HPLC Method:
		Flow Rate
	Time (min)	(μL/min)	A %	B %
	−0.5	350	100	0
	0.5	350	100	0
	1.0	350	10	90
	2.5	350	10	90
	2.51	350	100	10

TABLE 62
Tandem Mass Spectrometry
Ion Source       HESI-II
Polarity         Positive
SRM transitions
Tryptophan       205.1/118.2
Anthranilic acid 138.1/92.2
Tryptophan-d5    210.1/151.1

Example 44

Quantification of Tryptamine in Bacterial Supernatant by Liquid Chromatography-Mass Spectrometry (LC-MS)

Tryptamine acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μm/mL) were prepared. Samples (10 μL) and standards were mixed with 90 μL of ACN/H2O (60:30, v/v) containing 1 μg/mL of tryptamine-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.

LC-MS/MS Method

Tryptamine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 63., Table 64, and Table 65 provide the summary of the LC-MS/MS method.

TABLE 63
HPLC Method
Column           Accucore aQ column, 2.6 μm
                 (100 × 2.1 mm)
Mobile Phase A   99.9% H2O, 0.1% Formic Acid
Mobile Phase B   99.9% ACN, 0.1% Formic Acid
Injection volume 10 uL

TABLE 64
HPLC Method:
		Flow Rate
	Time (min)	(μL/min)	A %	B %
	−0.5	350	100	0
	0.5	350	100	0
	1.0	350	10	90
	2.5	350	10	90
	2.51	350	100	10

TABLE 65
Tandem Mass Spectrometry
Ion Source HESI-II
Polarity   Positive
SRM transitions
Tryptamine 161.1/144.1

Example 45

Quantification of Tryptophan, Indole-3-acetate, Indole-3-lactate, Indole-3-propionate in Bacterial Supernatant by High-pressure Liquid Chromatography (HPLC)

Samples were thawed on ice and centrifuged at 3,220×g for 5min at 4° C. 80 μL of the supernatant was pipetted, mixed with 20 μL 0.5% formic acid in water, and analyzed by HPLC using a Shimadzu Prominence-I. HPLC conditions used for the quantification of tryptophan, indole-3-acetate, indole-3-lactate and indole-3-propionate are described in Table 66.

TABLE 66
HPLC Analysis
Chromatography
Calibration standards	250, 100, 20, 4, 0.8 μg/mL
Column	Luna 3 μm C18(2) 100 Å, 100 × 2 mm (catalog#
	00D-4251-B0)
Column Temperature	40° C.
Injection Volume	10 μL
Autosampler	10° C.
Temperature
Flow Rate	0.5 mL/min
Mobile Phases	A: water, 0.1% FA
	B: acetonitrile, 0.1% FA
	Time (min)	% A	% B
Gradient	0	90	10
	0.5	90	10
	3	10	90
	5	10	90
	5.01	90	10
	7	(end)
Detection: Photodiode Array Detector (PDA)
Polarity	Positive
Start Wavelength	190 nm
End Wavelength	800 nm
Spectrum resolution	512
Slit Width	8 nm
Compound	Wavelength (nm)	Retention time (min)
Tryptophan	274	1.3
Indole-3-acetate	274	3.5
Indole-lactate	274	3.3
Indole-3-propionate	274	3.7

Example 46

Generation of Constructs for Overproducing Therapeutic Molecules for Secretion

To produce strain capable of secreting anti-inflammatory or gut barrier enhancer polypeptides, e.g., GLP2, IL-22, IL-10 (viral or human), several constructs are designed employing different secretion strategies. Various GLP2, IL-22, IL-10 (viral or human) constructs are synthesized, and cloned into vector pBR322 for transformation of E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome. In some embodiments, the constructs encoding the effector molecules are on a plasmid, e.g., a medium copy plasmid. Table 67 lists exemplary polypeptide coding sequences used in the constructs.

TABLE 67
Polypeptide coding sequences
Description	Sequence	SEQ ID NO
GLP2	CATGCTGATGGTTCTTTCTCTGATGAGAT	SEQ ID NO: 285
	GAACACCATTCTTGATAATCTTGCCGCCA
	GGGACTTTATAAACTGGTTGATTCAGACC
	AAAATCACTGAC
GLP2 codon	CATGCTGACGGCTCTTTTTCTGACGAAAT	SEQ ID NO: 286
optimized	GAATACCATCCTGGATAATCTGGCGGCG
	CGTGATTTTATTAATTGGCTGATCCAAAC
	TAAAATTACTGATTAA
FliC20-GLP2	ATGGCACAAGTCATTAATACCAACAGCC	SEQ ID NO: 287
(F1iC20, start of FliC	TCTCGCTGATCACTCAAAATAATATCAAC
gene preceding	AAGCATGCTGACGGCTCTTTTTCTGACGA
GLP2 sequence	AATGAATACCATCCTGGATAATCTGGCG
underlined)	GCGCGTGATTTTATTAATTGGCTGATCCA
	AACTAAAATTACTGATTAA
GLP2 codon	ATGCATGCTGACGGCTCTTTTTCTGACGA	SEQ ID NO: 288
optimized (e.g., used	AATGAATACCATCCTGGATAATCTGGCG
in fliC construct)	GCGCGTGATTTTATTAATTGGCTGATCCA
	AACTAAAATTACTGATTAA
vIL10 codon	ATGGGTACTGACCAATGTGATAATTTCCC	SEQ ID NO: 289
optimized (e.g., used	ACAAATGCTGCGTGATTTGCGCGACGCTT
in fliC construct)	TCTCGCGTGTGAAAACTTTTTTTCAGACT
	AAAGATGAGGTGGATAATCTGCTGCTGA
	AAGAGAGCCTGTTGGAAGATTTTAAAGG
	CTACTTGGGCTGTCAAGCGCTGTCGGAG
	ATGATTCAATTTTATCTGGAAGAGGTGAT
	GCCGCAAGCTGAGAACCAAGATCCGGAA
	GCGAAAGATCACGTGAATTCGCTGGGCG
	AGAATCTGAAAACTCTGCGTCTGCGTCTG
	CGTCGTTGTCACCGTTTTTTGCCGTGCGA
	AAACAAAAGTAAAGCTGTTGAGCAAATT
	AAAAACGCTTTTAACAAACTGCAGGAAA
	AAGGTATCTATAAAGCGATGAGCGAATT
	TGATATTTTTATTAATTATATTGAAGCTT
	ATATGACTATTAAAGCTCGCTAA
vIL10	GGTACAGACCAATGTGACAATTTTCCCCA	SEQ ID NO: 290
	AATGTTGAGGGACCTAAGAGATGCCTTC
	AGTCGTGTTAAAACCTTTTTCCAGACAAA
	GGACGAGGTAGATAACCTTTTGCTCAAG
	GAGTCTCTGCTAGAGGACTTTAAGGGCT
	ACCTTGGATGCCAGGCCCTGTCAGAAAT
	GATCCAATTCTACCTGGAGGAAGTCATG
	CCACAGGCTGAAAACCAGGACCCTGAAG
	CCAAAGACCATGTCAATTCTTTGGGTGAA
	AATCTAAAGACCCTACGGCTCCGCCTGC
	GCCGTTGCCACAGGTTCCTGCCGTGTGAG
	AACAAGAGTAAAGCTGTGGAACAGATAA
	AAAATGCCTTTAACAAGCTGCAGGAAAA
	AGGAATTTACAAAGCCATGAGTGAATTT
	GACATTTTTATTAACTACATAGAAGCATA
	CATGACAATTAAAGCCAGG
IL-22 codon	GCACCGATCTCTTCCCACTGTCGCTTAGA	SEQ ID NO: 291
optimized (e.g., use	TAAATCGAATTTTCAACAACCTTATATTA
with diffusible outer	CGAATCGTACGTTTATGCTGGCTAAAGA
membrane construct)	AGCGTCATTAGCTGATAACAACACTGAT
	GTTCGCCTGATTGGTGAGAAATTGTTTCA
	CGGTGTGTCTATGTCAGAACGTTGCTACC
	TGATGAAACAAGTTCTGAATTTCACCCTG
	GAAGAAGTGTTGTTTCCGCAATCTGACCG
	CTTTCAACCGTATATGCAAGAGGTTGTGC
	CGTTTCTGGCGCGCCTGAGTAATCGCCTG
	AGCACTTGTCATATTGAGGGCGACGACC
	TGCATATTCAACGAAATGTTCAAAAATTG
	AAAGATACGGTGAAGAAACTGGGTGAAA
	GTGGTGAAATCAAAGCGATTGGTGAGCT
	GGATCTGCTGTTTATGTCATTGCGCAATG
	CGTGCATTTAA
IL-22 codon	ATGGCACCGATCTCTTCCCACTGTCGCTT	SEQ ID NO: 292
optimized (e.g., used	AGATAAATCGAATTTTCAACAACCTTATA
in fliC construct)	TTACGAATCGTACGTTTATGCTGGCTAAA
	GAAGCGTCATTAGCTGATAACAACACTG
	ATGTTCGCCTGATTGGTGAGAAATTGTTT
	CACGGTGTGTCTATGTCAGAACGTTGCTA
	CCTGATGAAACAAGTTCTGAATTTCACCC
	TGGAAGAAGTGTTGTTTCCGCAATCTGAC
	CGCTTTCAACCGTATATGCAAGAGGTTGT
	GCCGTTTCTGGCGCGCCTGAGTAATCGCC
	TGAGCACTTGTCATATTGAGGGCGACGA
	CCTGCATATTCAACGAAATGTTCAAAAAT
	TGAAAGATACGGTGAAGAAACTGGGTGA
	AAGTGGTGAAATCAAAGCGATTGGTGAG
	CTGGATCTGCTGTTTATGTCATTGCGCAA
	TGCGTGCATTTAA
hIL-10 codon	TCGCCAGGTCAAGGAACGCAGTCAGAGA	SEQ ID NO: 293
optimized	ATTCATGCACTCACTTTCCGGGCAATCTG
	CCGAATATGCTGCGCGATCTGCGAGATG
	CATTCTCTCGCGTGAAAACGTTCTTTCAA
	ATGAAAGATCAACTGGATAATCTGCTGC
	TGAAGGAGTCGTTGTTGGAGGATTTTAA
	GGGGTATCTGGGTTGTCAAGCACTGTCTG
	AAATGATTCAATTTTACTTGGAGGAAGTT
	ATGCCGCAAGCGGAAAACCAAGATCCGG
	ATATTAAGGCGCACGTGAACTCACTGGG
	CGAAAACCTGAAAACTTTGCGCCTGCGT
	CTGAGACGATGTCACCGATTCCTGCCGTG
	TGAAAACAAGTCAAAGGCGGTTGAGCAA
	GTTAAGAATGCTTTCAATAAGCTGCAAG
	AAAAGGGCATCTATAAAGCGATGTCTGA
	ATTTGATATCTTTATAAACTACATAGAAG
	CTTATATGACTATGAAGATTCGAAATTAA
Monomerized hIL-	TCGCCAGGTCAAGGAACGCAGTCAGAGA	SEQ ID NO: 294
10 (codon opt)	ATTCATGCACTCACTTTCCGGGCAATCTG
	CCGAATATGCTGCGCGATCTGCGAGATG
	CATTCTCTCGCGTGAAAACGTTCTTTCAA
	ATGAAAGATCAACTGGATAATCTGCTGC
	TGAAGGAGTCGTTGTTGGAGGATTTTAA
	GGGGTATCTGGGTTGTCAAGCACTGTCTG
	AAATGATTCAATTTTACTTGGAGGAAGTT
	ATGCCGCAAGCGGAAAACCAAGATCCGG
	ATATTAAGGCGCACGTGAACTCACTGGG
	CGAAAACCTGAAAACTTTGCGCCTGCGT
	CTGAGACGATGTCACCGATTCCTGCCGTG
	TGAAAACGGAGGAGGAAGTGGTGGTAAG
	TCAAAGGCGGTTGAGCAAGTTAAGAATG
	CTTTCAATAAGCTGCAAGAAAAGGGCAT
	CTATAAAGCGATGTCTGAATTTGATATCT
	TTATAAACTACATAGAAGCTTATATGACT
	ATGAAGATTCGAAATTAA

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, or SEQ ID NO: 294 or a functional fragment thereof.

Table 68 lists exemplary secretion tags, which can be added at the N-terminus when the diffusible outer membrane (DOM) method or the fliC method is used.

TABLE 68
Secretion Tags and FliC components
Sequence Name	Sequence	SEQ ID NO
fliC-FliC20 (e.g., used in GLP2	tgacggcgattgagccgacgggtggaaaccc	SEQ ID NO: 295
construct)	aaaacgtaatcaac GTGGGTACTC
FliC20: start of the fliC gene	CTTAAATTGGGTTCGAATGG
which (in some constructs)	ACC atggcacaagtcattaataccaacagc
precedes the effector polypeptide	ctctcgctgatcactcaaaataatatcaacaag
sequence, see e.g., FIG. 30B and
FIG. 30C shown in italics
fliC: native fliC UTR in bold,
optimized RBS underlined
fliC-RBS (e.g., used in IL22	tgacggcgattgagccgacgggtggaaaccc	SEQ ID NO: 296
construct)	aaaacgtaatcaac tacgaacacttacagga
fliC: native fliC UTR in bold,	ggtaccca
optimized RBS underlined
fliC-RBS (e.g., used in GLP2	tgacggcgattgagccgacgggtggaaaccc	SEQ ID NO: 297
construct)	aaaacgtaatcaac aagtataaactctggga
fliC: native fliC UTR in bold,	ggttccta
optimized RBS underlined
fliC-RBS (e.g., used in vIL10	tgacggcgattgagccgacgggtggaaaccc	SEQ ID NO: 298
construct)	aaaacgtaatcaac tcaaatcc c ttaataagg
fliC: native fliC UTR in bold,	aggtaaa
optimized RBS underlined
RBS-phoA	Ctctagaaataattttgtttaactttaagaaggaga	SEQ ID NO: 299
RBS: underlined	tatacatatgaaacaaagcactattgcactggca
	ctcttaccgttactgtttacccctgtgacaaaagc
	g
phoA	atgaaacaaagcactattgcactggcactcttac	SEQ ID NO: 300
	cgttactgtttacccctgtgacaaaagcg
RBS-ompF	Ctctagaaataattttgtttaactttaagaaggaga	SEQ ID NO: 301
RBS: underlined	tatacatatgatgaagcgcaatattctggcagtga
	tcgtccctgctctgttagtagcaggtactgcaaac
	gct
ompF	atgatgaagcgcaatattctggcagtgatcgtcc	SEQ ID NO: 302
	ctgctctgttagtagcaggtactgcaaacgct
RBS-cvaC	Ctctagaaataattttgtttaactttaagaaggaga	SEQ ID NO: 303
RBS: underlined	tatacatATGAGAACTCTGACTCT
	AAATGAATTAGATTCTGTTTC
	TGGTGGT
cvaC	ATGAGAACTCTGACTCTAAAT	SEQ ID NO: 304
	GAATTAGATTCTGTTTCTGGT
	GGT
RBS-phoA (Optimized, e.g.,	GACGCCAGAGAGTTAAGGGG	SEQ ID NO: 305
used in IL10 construct)	GTTAAATGAAACAATCGACC
RBS: underlined	ATCGCATTGGCGCTGCTTCCT
	CTATTGTTCACACCGGTGACA
	AAGGCA
Optimized phoA	ATGAAACAATCGACCATCGC	SEQ ID NO: 306
	ATTGGCGCTGCTTCCTCTATT
	GTTCACACCGGTGACAAAGG
	CA
RBS-TorA	ctctagaaataattttgtttaactttaagaaggagat	SEQ ID NO: 307
RBS: underlined	atacatATGAACAATAACGATCT
	CTTTCAGGCATCACGTCGGCG
	TTTTCTGGCACAACTCGGCGG
	CTTAACCGTCGCCGGGATGCT
	GGGGCCGTCATTGTTAACGCC
	GCGACGTGCGACTGCG
TorA	ATGAACAATAACGATCTCTTT	SEQ ID NO: 308
	CAGGCATCACGTCGGCGTTTT
	CTGGCACAACTCGGCGGCTTA
	ACCGTCGCCGGGATGCTGGG
	GCCGTCATTGTTAACGCCGCG
	ACGTGCGACTGCG
RBS-TorA alternate	CCCACATTCGAGGTACTAAatg	SEQ ID NO: 309
	aacaataacgatctctttcaggcatcacgtcggc
	gttttctggcacaactcggcggcttaaccgtcgc
	cgggatgctggggacgtcattgttaacgccgcg
	ccgtgcgactgcggcgcaagcggcg
TorA (alternate)	atgaacaataacgatctctttcaggcatcacgtcg	SEQ ID NO: 310
	gcgttttctggcacaactcggcggcttaaccgtc
	gccgggatgctggggacgtcattgttaacgccg
	cgccgtgcgactgcggcgcaagcggcg
RBS-fdnG	ACCCTATTACACACCTAAGGA	SEQ ID NO: 311
	GGCCAAATACatggacgtcagtcgcag
	acaattttttaaaatctgcgcgggcggtatggcg
	ggaacaacagtagcagcattgggctttgccccg
	aagcaagcactggct
fdnG	atggacgtcagtcgcagacaattttttaaaatctg	SEQ ID NO: 312
	cgcgggcggtatggcgggaacaacagtagca
	gcattgggctttgccccgaagcaagcactggct
RBS-dmsA	TACGCAAAAAACATAATTTAA	SEQ ID NO: 313
	GAGAGGATAAACatgaaaacgaaaa
	tccctgatgcggtattggctgctgaggtgagtcg
	ccgtggtttggtaaaaacgacagcgatcggcgg
	cctggcaatggccagcagcgcattaacattacct
	tttagtcggattgcgcacgct
dmsA	atgaaaacgaaaatccctgatgcggtattggctg	SEQ ID NO: 314
	ctgaggtgagtcgccgtggtttggtaaaaacgac
	agcgatcggcggcctggcaatggccagcagcg
	cattaacattaccttttagtcggattgcgcacgct

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, and SEQ ID NO: 313. Table 69 lists exemplary promoter sequences and miscellaneous construct sequences.

TABLE 69
Promoter Sequences and Various Construct Sequences
		SEQ ID
Description	Sequence	NO
TetR/TetA	gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaag	SEQ ID
Promoter	gccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaata	NO: 315
	atggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgacttgatc
	ttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctct
	agtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttct
	gtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaa
	acttttagcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgag
	tatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacat
	gccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaacctt
	cgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctaga
	catcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatc
	agtgatagagaaaagtgaa
fliC Promoter	agcgggaataaggggcagagaaaagagtatttcgtcgactaacaaaaaatggctgtttgt	SEQ ID
	gaaaaaaattctaaaggttgttttacgacagacgataacagggt	NO: 316
FnrS	ggtaccAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT	SEQ ID
Promoter	AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAA	NO: 317
	CGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGG
	CAATATCTCTCTTggatcc
DOM	cacatttccccgaaaagtgccgatggccccccgatggtagtgtggcccatgcgagagtagg	SEQ ID
Construct	gaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttat	NO: 318
Terminator	ctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacg
	ttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcat
	caaattaagcagaaggccatcctgacggatggcctttttgcgtggccagtgccaagcttgc
	atgcagattgcagcattacacgtcttgagcgattgtgtaggctggagctgcttc
FRT Site	gaagttcctatactttctagagaataggaacttcggaataggaacttc	SEQ ID
		NO: 319
Kanamycin	aagatcccctcacgctgccgcaagcactcagggcgcaagggctgctaaaggaagcggaa	SEQ ID
Resistance	cacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggc	NO: 320
Cassette (for	tatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggctta
integration	catggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctg
in between	gggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgcc
FRT sites)	aaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcg
	catgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattc
	ggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcag
	cgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcag
	gacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcg
	acgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatc
	tcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg
	ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagc
	gagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc
	aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgag
	gatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgctt
	ttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttgg
	ctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttac
	ggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga
	gcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt
	tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggc
	tggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccagcttcaaaag
	cgctct

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, and SEQ ID NO: 320. Table 70 Lists exemplary secretion constructs.

TABLE 70
Non-limiting Examples of Secretion Constructs
Description	Sequence	SEQ ID NO:
FliC20-glp2; a human	cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc	SEQ ID NO:
GLP2 construct	gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct	321
inserted into the FliC	gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag
locus, under the	cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca
control of the native	gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtattaat
FliC promoter	cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt
	gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa
	attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa
	aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag
	cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg
	attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc
	agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG
	cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc
	gtcggctcaatcgccgtcaaccctgttatcgtctgtcgtaaaacaacctttag
	aatttttttcacaaacagccattttttgttagtcgacgaaatactcttttctctgc
	cccttattcccgctattaaaaaaaacaattaaacgtaaactttgcgcaattca
	ggccgataaccccggtattcgttttacgtgtcgaaagataaaCGAAGT
	TCCTATACTTTCTAGAGAATAGGAACTTCGG
	AATAGGAACTTCATTTctcgttcgctgccacctaagaatact
	ctacggtcacatacAAATGGCGCGCCTTACGCCCCGC
	CCTGCCACTCATCGCAGTACTGTTGTATTCAT
	TAAGCATCTGCCGACATGGAAGCCATCACAA
	ACGGCATGATGAACCTGAATCGCCAGCGGCA
	TCAGCACCTTGTCGCCTTGCGTATAATATTTG
	CCCATGGTGAAAACGGGGGCGAAGAAGTTGT
	CCATATTGGCCACGTTTAAATCAAAACTGGT
	GAAACTCACCCAGGGATTGGCTGAGACGAAA
	AACATATTCTCAATAAACCCTTTAGGGAAAT
	AGGCCAGGTTTTCACCGTAACACGCCACATC
	TTGCGAATATATGTGTAGAAACTGCCGGAAA
	TCGTCGTGGTATTCACTCCAGAGCGATGAAA
	ACGTTTCAGTTTGCTCATGGAAAACGGTGTA
	ACAAGGGTGAACACTATCCCATATCACCAGC
	TCACCGTCTTTCATTGCCATACGTAATTCCGG
	ATGAGCATTCATCAGGCGGGCAAGAATGTGA
	ATAAAGGCCGGATAAAACTTGTGCTTATTTTT
	CTTTACGGTCTTTAAAAAGGCCGTAATATCC
	AGCTGAACGGTCTGGTTATAGGTACATTGAG
	CAACTGACTGAAATGCCTCAAAATGTTCTTT
	ACGATGCCATTGGGATATATCAACGGTGGTA
	TATCCAGTGATTTTTTTCTCCATTTTAGCTTCC
	TTAGCTCCTGAAAATCTCGACAACTCAAAAA
	ATACGCCCGGTAGTGATCTTATTTCATTATGG
	TGAAAGTTGGAACCTCTTACGTGCCGATCAA
	CGTCTCATTTTCGCCAAAAGTTGGCCCAGGG
	CTTCCCGGTATCAACAGGGACACCAGGATTT
	ATTTATTCTGCGAAGTGATCTTCCGTCACAGG
	TAGGCGCGCCGAAGTTCCTATACTTTCTAGA
	GAATAGGAACTTCGGAATAGGAACTctcaccgcc
	gcgcaaaaagcgacgctaacccctatttcaaatcagcaatcgtcgtttacc
	gctaaacttagcgcctacggtacgctgaaaagcgcgctgacgactttcca
	gaccgccaatactgcattgtctaaagccgatcttttttccgctaccagcacc
	accagcagcaccaccgcgttcagtgccaccaccgcgggtaatgccatcg
	ccgggaaatacaccatcagcgtcacccatctggcgcaggcgcaaaccct
	gacaacgcgcaccaccagagacgatacgaaaacggcgatcgccacca
	gcgacagcaaactcaccattcaacaaggcggcgacaaagatccgatttcc
	attgatatcagcgcggctaactcgtctttaagcgggatccgtgatgccatca
	acaacgcaaaagcaggcgtaagcgcaagcatcattaacgtgggtaacgg
	tgaatatcgtctgtcagtcacatcaaatgacaccggcct
FliC20 with optimized	attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc	SEQ ID NO:
RBS-GLP2 and UTR-	agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG	322
FliC	cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc
	gtcggctcaatcgccgtca
human GLP2	cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc	SEQ ID NO:
construct,, including	gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct	323
the N terminal 20	gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag
amino acids of FliC	cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca
(reverse orientation),	gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtattaat
inserted into the FliC	cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt
locus under the control	gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa
of a tet inducible	attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa
promoter, with TetR	aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag
and chloramphenicol	cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg
resistance.	attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc
	agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG
	cttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgt
	gccatGGTCCATTCGAACCCAATTTAAGGAGTA
	CCCACgttgattacgttttgggtttccacccgtcggctcaatcgccgtca
	ttctctatcactgatagggagtggtaaaataactctatcaatgatagagtgtc
	aacaaaaattaggaattaatgatgtctagattagataaaagtaaagtgattaa
	cagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccg
	taaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgt
	aaaaaataagcgggctttgctcgacgccttagccattgagatgttagatag
	gcaccatactcacttttgccctttagaaggggaaagctggcaagattttttac
	gtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagca
	aaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaa
	aatcaattagccatttatgccaacaaggtttttcactagagaatgcattatatg
	cactcagcgctgtggggcattttactttaggttgcgtattggaagatcaaga
	gcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgc
	cgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagcc
	agccttcttattcggccttgaattgatcatatgcggattagaaaaacaactta
	aatgtgaaagtgggtcttaagaattatttcacaaacagccattattgttagtc
	gacgaaatactcttttctctgccccttattcccgctattaaaaaaaacaattaa
	acgtaaactttgcgcaattcaggccgataaccccggtattcgttttacgtgtc
	gaaagataaaCGAAGTTCCTATACTTTCTAGAGAA
	TAGGAACTTCGGAATAGGAACTTCATTTctcgtt
	cgctgccacctaagaatactctacggtcacatacAAATGGCGCG
	CCTTACGCCCCGCCCTGCCACTCATCGCAGTA
	CTGTTGTATTCATTAAGCATCTGCCGACATGG
	AAGCCATCACAAACGGCATGATGAACCTGAA
	TCGCCAGCGGCATCAGCACCTTGTCGCCTTG
	CGTATAATATTTGCCCATGGTGAAAACGGGG
	GCGAAGAAGTTGTCCATATTGGCCACGTTTA
	AATCAAAACTGGTGAAACTCACCCAGGGATT
	GGCTGAGACGAAAAACATATTCTCAATAAAC
	CCTTTAGGGAAATAGGCCAGGTTTTCACCGT
	AACACGCCACATCTTGCGAATATATGTGTAG
	AAACTGCCGGAAATCGTCGTGGTATTCACTC
	CAGAGCGATGAAAACGTTTCAGTTTGCTCAT
	GGAAAACGGTGTAACAAGGGTGAACACTATC
	CCATATCACCAGCTCACCGTCTTTCATTGCCA
	TACGTAATTCCGGATGAGCATTCATCAGGCG
	GGCAAGAATGTGAATAAAGGCCGGATAAAA
	CTTGTGCTTATTTTTCTTTACGGTCTTTAAAA
	AGGCCGTAATATCCAGCTGAACGGTCTGGTT
	ATAGGTACATTGAGCAACTGACTGAAATGCC
	TCAAAATGTTCTTTACGATGCCATTGGGATAT
	ATCAACGGTGGTATATCCAGTGATTTTTTTCT
	CCATTTTAGCTTCCTTAGCTCCTGAAAATCTC
	GACAACTCAAAAAATACGCCCGGTAGTGATC
	TTATTTCATTATGGTGAAAGTTGGAACCTCTT
	ACGTGCCGATCAACGTCTCATTTTCGCCAAA
	AGTTGGCCCAGGGCTTCCCGGTATCAACAGG
	GACACCAGGATTTATTTATTCTGCGAAGTGA
	TCTTCCGTCACAGGTAGGCGCGCCGAAGTTC
	CTATACTTTCTAGAGAATAGGAACTTCGGAA
	TAGGAACTctcaccgccgcgcaaaaagcgacgctaacccctattt
	caaatcagcaatcgtcgtttaccgctaaacttagcgcctacggtacgctga
	aaagcgcgctgacgactttccagaccgccaatactgcattgtctaaagccg
	atcttttttccgctaccagcaccaccagcagcaccaccgcgttcagtgcca
	ccaccgcgggtaatgccatcgccgggaaatacaccatcagcgtcaccca
	tctggcgcaggcgcaaaccctgacaacgcgcaccaccagagacgatac
	gaaaacggcgatcgccaccagcgacagcaaactcaccattcaacaagg
	cggcgacaaagatccgatttccattgatatcagcgcggctaactcgtcttta
	agcgggatccgtgatgccatcaacaacgcaaaagcaggcgtaagcgca
	agcatcattaacgtgggtaacggtgaatatcgtctgtcagtcacatcaaatg
	acaccggcct
human GLP2	ttaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcca	SEQ ID NO:
construct,, including	gattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATGc	324
the N terminal 20	ttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgtg
amino acids of FliC	ccat
(reverse orientation)
human GLP2	ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaa	SEQ ID NO:
construct with a N	ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag	325
terminal OmpF	cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt
secretion tag (sec-	agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc
dependent secretion	acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata
system) under the	aaaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacc
control of a tet	taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt
inducible promoter,	agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt
includes TetR in	gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc
reverse direction	gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg
	cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat
	gcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt
	gttgacactctatcattgatagagttattttaccactccctatcagtgatagagaa
	aagtgaactctagaaataattttgtttaactttaagaaggagatatacatatga
	tgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcaggtac
	tgcaaacgctcatgctgatggttctttctctgatgagatgaacaccattcttga
	taatcttgccgccagggactttataaactggttgattcagaccaaaatcactg
	acaggtgacacatttccccgaaaagtgccgatggccccccgatggtagtg
	tggccccatgcgagagtagggaactgccaggcatcaaataaaacgaaag
	gctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct
	ctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca
	acggcccggagggtggcgggcaggacgcccgccataaactgccaggc
	atcaaattaagcagaaggccatcctgacggatggcctttttgcgtggccag
	tgccaagcttgcatgcagattgcagcattacacgtcttgagcgattgtgtag
	gctggagctgcttcgaagttcctatactttctagagaataggaacttcggaat
	aggaacttc
human GLP2	atgatgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcag	SEQ ID NO:
construct with a N	gtactgcaaacgctcatgctgatggttctttctctgatgagatgaacaccatt	326
terminal OmpF	cttgataatcttgccgccagggactttataaactggttgattcagaccaaaat
secretion tag (sec-	cactgacaggtga
dependent secretion
system)
human GLP2	taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaa	SEQ ID NO:
construct with a N	ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag	327
terminal TorA	cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt
secretion tag (tat	agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc
secretion system)	acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata
under the control of	aaaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacc
a tet inducible	taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt
promoter	agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt
	gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc
	gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg
	cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat
	gcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt
	gttgacactctatcattgatagagttattttaccactccctatcagtgatagagaa
	aagtgaactctagaaataattttgtttaactttaagaaggagatatacatAT
	GAACAATAACGATCTCTTTCAGGCATCACGT
	CGGCGTTTTCTGGCACAACTCGGCGGCTTAA
	CCGTCGCCGGGATGCTGGGGCCGTCATTGTT
	AACGCCGCGACGTGCGACTGCGcatgctgatggttctt
	tctctgatgagatgaacaccattcttgataatcttgccgccagggactttata
	aactggttgattcagaccaaaatcactgactaataacacatttccccgaaaa
	gtgccgatggccccccgatggtagtgtggcccatgcgagagtagggaac
	tgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttc
	gttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccg
	ggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggca
	ggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcc
	tgacggatggcctttttgcgtggccagtgccaagcttgcatgcagattgca
	gcattacacgtcttgagcgattgtgtaggctggagctgcttcgaagttcctat
	actttctagagaataggaacttcggaataggaacttc
GLP-2 with TORA tag	ATGAACAATAACGATCTCTTTCAGGCATCAC	SEQ ID NO:
	GTCGGCGTTTTCTGGCACAACTCGGCGGCTT	328
	AACCGTCGCCGGGATGCTGGGGCCGTCATTG
	TTAACGCCGCGACGTGCGACTGCGcatgctgatggt
	tctttctctgatgagatgaacaccattcttgataatcttgccgccagggacttt
	ataaactggttgattcagaccaaaatcactgac

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQ ID NO: 328. Table 71 lists exemplary secretion constructs.

TABLE 71
Non-limiting Examples of Secretion Constructs
Description	Sequences	SEQ ID NO
Ptet-phoA-hIL10	gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat	SEQ ID NO:
	gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca	329
	aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg
	tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac
	ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt
	gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata
	ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga
	cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc
	cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc
	tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac
	aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa
	ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac
	ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt
	gatagagttattttaccactccctatcagtgatagagaaaagtgaa
	GACGCCAGAGAGTTAAGGGGGTTAAATGAA
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC
	TATTGTTCACACCGGTGACAAAGGCA
	TCGCCAGGTCAAGGAACGCAGTCAGAGAATT
	CATGCACTCACTTTCCGGGCAATCTGCCGAA
	TATGCTGCGCGATCTGCGAGATGCATTCTCTC
	GCGTGAAAACGTTCTTTCAAATGAAAGATCA
	ACTGGATAATCTGCTGCTGAAGGAGTCGTTG
	TTGGAGGATTTTAAGGGGTATCTGGGTTGTC
	AAGCACTGTCTGAAATGATTCAATTTTACTTG
	GAGGAAGTTATGCCGCAAGCGGAAAACCAA
	GATCCGGATATTAAGGCGCACGTGAACTCAC
	TGGGCGAAAACCTGAAAACTTTGCGCCTGCG
	TCTGAGACGATGTCACCGATTCCTGCCGTGT
	GAAAACAAGTCAAAGGCGGTTGAGCAAGTT
	AAGAATGCTTTCAATAAGCTGCAAGAAAAGG
	GCATCTATAAAGCGATGTCTGAATTTGATAT
	CTTTATAAACTACATAGAAGCTTATATGACT
	ATGAAGATTCGAAATTAA
phoA-hIL10	GACGCCAGAGAGTTAAGGGGGTTAAATGAA	SEQ ID NO:
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC	330
	TATTGTTCACACCGGTGACAAAGGCA
	TCGCCAGGTCAAGGAACGCAGTCAGAGAATT
	CATGCACTCACTTTCCGGGCAATCTGCCGAA
	TATGCTGCGCGATCTGCGAGATGCATTCTCTC
	GCGTGAAAACGTTCTTTCAAATGAAAGATCA
	ACTGGATAATCTGCTGCTGAAGGAGTCGTTG
	TTGGAGGATTTTAAGGGGTATCTGGGTTGTC
	AAGCACTGTCTGAAATGATTCAATTTTACTTG
	GAGGAAGTTATGCCGCAAGCGGAAAACCAA
	GATCCGGATATTAAGGCGCACGTGAACTCAC
	TGGGCGAAAACCTGAAAACTTTGCGCCTGCG
	TCTGAGACGATGTCACCGATTCCTGCCGTGT
	GAAAACAAGTCAAAGGCGGTTGAGCAAGTT
	AAGAATGCTTTCAATAAGCTGCAAGAAAAGG
	GCATCTATAAAGCGATGTCTGAATTTGATAT
	CTTTATAAACTACATAGAAGCTTATATGACT
	ATGAAGATTCGAAATTAA
fliC UTR-RBS-	tgacggcgattgagccgacgggtggaaacccaaaacgtaatcaac t	SEQ ID NO:
pvIL10	caaatcccttaataaggaggtaaa ATGGGTACTGACCAA	331
	TGTGATAATTTCCCACAAATGCTGCGTGATTT
	GCGCGACGCTTTCTCGCGTGTGAAAACTTTTT
	TTCAGACTAAAGATGAGGTGGATAATCTGCT
	GCTGAAAGAGAGCCTGTTGGAAGATTTTAAA
	GGCTACTTGGGCTGTCAAGCGCTGTCGGAGA
	TGATTCAATTTTATCTGGAAGAGGTGATGCC
	GCAAGCTGAGAACCAAGATCCGGAAGCGAA
	AGATCACGTGAATTCGCTGGGCGAGAATCTG
	AAAACTCTGCGTCTGCGTCTGCGTCGTTGTCA
	CCGTTTTTTGCCGTGCGAAAACAAAAGTAAA
	GCTGTTGAGCAAATTAAAAACGCTTTTAACA
	AACTGCAGGAAAAAGGTATCTATAAAGCGAT
	GAGCGAATTTGATATTTTTATTAATTATATTG
	AAGCTTATATGACTATTAAAGCTCGCTAA
Ptet-phoA-vIL10	Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat	SEQ ID NO:
	gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca	332
	aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg
	tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac
	ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt
	gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata
	ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga
	cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc
	cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc
	tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac
	aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa
	ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac
	ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt
	gatagagttattttaccactccctatcagtgatagagaaaagtgaa
	GACGCCAGAGAGTTAAGGGGGTTAAATGAA
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC
	TATTGTTCACACCGGTGACAAAGGCA
	GGTACAGACCAATGTGACAATTTTCCCCAAA
	TGTTGAGGGACCTAAGAGATGCCTTCAGTCG
	TGTTAAAACCTTTTTCCAGACAAAGGACGAG
	GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT
	AGAGGACTTTAAGGGCTACCTTGGATGCCAG
	GCCCTGTCAGAAATGATCCAATTCTACCTGG
	AGGAAGTCATGCCACAGGCTGAAAACCAGG
	ACCCTGAAGCCAAAGACCATGTCAATTCTTT
	GGGTGAAAATCTAAAGACCCTACGGCTCCGC
	CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG
	AGAACAAGAGTAAAGCTGTGGAACAGATAA
	AAAATGCCTTTAACAAGCTGCAGGAAAAAGG
	AATTTACAAAGCCATGAGTGAATTTGACATT
	TTTATTAACTACATAGAAGCATACATGACAA
	TTAAAGCCAGG
phoA-vIL10	GACGCCAGAGAGTTAAGGGGGTTAAATGAA	SEQ ID NO:
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC	333
	TATTGTTCACACCGGTGACAAAGGCA
	GGTACAGACCAATGTGACAATTTTCCCCAAA
	TGTTGAGGGACCTAAGAGATGCCTTCAGTCG
	TGTTAAAACCTTTTTCCAGACAAAGGACGAG
	GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT
	AGAGGACTTTAAGGGCTACCTTGGATGCCAG
	GCCCTGTCAGAAATGATCCAATTCTACCTGG
	AGGAAGTCATGCCACAGGCTGAAAACCAGG
	ACCCTGAAGCCAAAGACCATGTCAATTCTTT
	GGGTGAAAATCTAAAGACCCTACGGCTCCGC
	CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG
	AGAACAAGAGTAAAGCTGTGGAACAGATAA
	AAAATGCCTTTAACAAGCTGCAGGAAAAAGG
	AATTTACAAAGCCATGAGTGAATTTGACATT
	TTTATTAACTACATAGAAGCATACATGACAA
	TTAAAGCCAGG
Ptet-PhoA-IL22	Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat	SEQ ID NO:
	gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca	334
	aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg
	tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac
	ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt
	gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata
	ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga
	cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc
	cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc
	tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac
	aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa
	ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac
	ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt
	gatagagttattttaccactccctatcagtgatagagaaaagtgaa
	GACGCCAGAGAGTTAAGGGGGTTAAATGAA
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC
	TATTGTTCACACCGGTGACAAAGGCA
	GCACCGATCTCTTCCCACTGTCGCTTAGATAA
	ATCGAATTTTCAACAACCTTATATTACGAATC
	GTACGTTTATGCTGGCTAAAGAAGCGTCATT
	AGCTGATAACAACACTGATGTTCGCCTGATT
	GGTGAGAAATTGTTTCACGGTGTGTCTATGTC
	AGAACGTTGCTACCTGATGAAACAAGTTCTG
	AATTTCACCCTGGAAGAAGTGTTGTTTCCGC
	AATCTGACCGCTTTCAACCGTATATGCAAGA
	GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC
	GCCTGAGCACTTGTCATATTGAGGGCGACGA
	CCTGCATATTCAACGAAATGTTCAAAAATTG
	AAAGATACGGTGAAGAAACTGGGTGAAAGT
	GGTGAAATCAAAGCGATTGGTGAGCTGGATC
	TGCTGTTTATGTCATTGCGCAATGCGTGCATT
	TAA
PhoA-IL22	GACGCCAGAGAGTTAAGGGGGTTAAATGAA	SEQ ID NO:
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC	335
	TATTGTTCACACCGGTGACAAAGGCA
	GCACCGATCTCTTCCCACTGTCGCTTAGATAA
	ATCGAATTTTCAACAACCTTATATTACGAATC
	GTACGTTTATGCTGGCTAAAGAAGCGTCATT
	AGCTGATAACAACACTGATGTTCGCCTGATT
	GGTGAGAAATTGTTTCACGGTGTGTCTATGTC
	AGAACGTTGCTACCTGATGAAACAAGTTCTG
	AATTTCACCCTGGAAGAAGTGTTGTTTCCGC
	AATCTGACCGCTTTCAACCGTATATGCAAGA
	GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC
	GCCTGAGCACTTGTCATATTGAGGGCGACGA
	CCTGCATATTCAACGAAATGTTCAAAAATTG
	AAAGATACGGTGAAGAAACTGGGTGAAAGT
	GGTGAAATCAAAGCGATTGGTGAGCTGGATC
	TGCTGTTTATGTCATTGCGCAATGCGTGCATT
	TAA
	GACGCCAGAGAGTTAAGGGGGTTAAATGAA	SEQ ID NO:
	ACAATCGACCATCGCATTGGCGCTGCTTCCTC	336
	TATTGTTCACACCGGTGACAAAGGCA

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, and SEQ ID NO: 336.

Example 47

Bacterial Secretion of hIL-10 and vIL-10

To determine whether the human IL-10 and vIL-10 expressed by engineered bacteria is secreted, the concentration of IL-10 in the bacterial supernatant from a selection of engineered strains comprising various hIL-10 and vIL-10 constructs/strains was measured (see Table 62, Table 63, Table 64, Table 65, Table 66 for components and sequences for hIL-10 and vIL-10 constructs/strains).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing.

The concentration of hIL-10 in the cell-free medium and in the bacterial cell extract was measured by hIL-10 ELISA (R&D Systems DY217B), according to manufacturer's instructions Similarly, to determine the concentrations of vIL-10 an Ultrasensitive ELISA kit (Alpco, 45-I10HUU-E01) was employed using commercially available recombinant vIL-10 (R&D Systems, 915-VL-010). All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-10. Standard curves were generated using both human and viral recombinant proteins. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 72 and Table 73 summarize levels of hIL10 and vIL-10 measured in the supernatant. The data show that both vIL-10 and hIL-10 are secreted at various levels from the different bacterial strains.

TABLE 72
hIL-10 Secretion
                        hu IL-10
                        (ng/ml)
Sample                  (extracellular)
WT                      0
IL-10 Plasmid (Nissle   8.4
pUC57.Ptet-phoA-hIL10)
IL-10 plasmid/lpp       19.3
(lpp::Cm pUC57.Ptet-phoA-
hIL10)
2083 IL-10 plasmid/nlpI 20.5
(nlpI::Cm pUC57.Ptet-phoA-
hIL10)
2084 IL-10 plasmid/tolA 21.4
(tolA::Cm pUC57.Ptet-phoA-
hIL10)
2085 IL-10 plasmid/pal  28.4
(PAL::Cm pUC57.Ptet-phoA-
hIL10)

TABLE 73
vIL-10 Secretion
                         vIL-10
                         (ng/ml)
Sample                   (extracellular)
WT                       0
fliC-pvIL10 (Nissle      29
pUN fli-vIL10 Kan Cm)
fliC::vIL10 (Nissle      9
fliC::vIL10 delta fliD CmR)
vIL-10 lpp (Nissle lpp   527
mutant with vIL10 pBR3222
tet plasmid)
vIL-10 nlp1 (Nissle      982
delta nlpI::CmR pBR322.Ptet-
phoA-vIL10)
vIL-10 tolA (Nissle      428
delta tolA::CmR pBR322.Ptet-
phoA-vIL10)
vIL-10 pal (Nissle delta 1090
PAL::CmR pBR322.Ptet-
phoA-vIL10

Co-Culture Studies

To determine whether the hIL-10 and viral IL-10 expressed by the genetically engineered bacteria shown in Table 72 and Table 73 is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant containing secreted human or viral IL-10 is added to the growth medium of a Raji cells (a hematopoietic cell line) and J774a1 cells (a macrophage cell line). IL-10 is known to induce the phosphorylation of STAT3 in these cells Functional activity of bacterially secreted IL-10 is therefore assessed by its ability to phosphorylate STAT3 in Raji and J774a1 cells.

Raji cells are grown in RPM! 1640 supplemented with 10% FBS supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, RPMI 1640 supplemented with 10% FBS (1e6/24 well) are serum starved overnight. Titrations of either recombinant human IL-10 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for 30 minutes. Cells are harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) is run in triplicate for all samples, according to manufacturer's instructions. PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity.

Competition Studies

As an additional control for specificity, a competition assay is performed. Titrations of anti-IL10 antibody are pre-incubated with constant concentrations of either rhIL22 (data not shown) or supernatants from the engineered bacteria expressing human or viral IL-22 for 15 min. Next, the supernatants/ rhIL2 solutions are added to serum-starved Raji cells (1e6/well) and cells are incubated for 30 min followed by pSTAT3 ELISA as described above.

In other embodiments, similar studies are conducted with J774a1 cells.

Example 48

Bacterial Secretion of GLP-2

To determine whether the human GLP-2 expressed by engineered bacteria is secreted, the concentration of GLP-2 in the bacterial supernatant from two engineered strains comprising GLP-2 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing GLP-2 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing GLP-2, further comprising a deletion in degP (see Table 74).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing.

The concentration of GLP-2 in the cell-free medium and in the bacterial cell extract was measured by Human GLP2 ELISA Kit (Competitive EIA) (LSBio), according to manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of GLP-2. Standard curves were generated using recombinant GLP-2. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. As seen in Table 74, deletion of degP, a periplasmic protease, improved secretion levels over 3-fold.

TABLE 74
GLP-2 Secretion
DOM mut                  ng/ml
WT                       1.14
PAL::CmR Ptet-ompF-GLP2  1793.2
PAL::CmR ompT::Kan Ptet- 1142.1
ompF-GLP2
PAL::CmR ompT::Kan phoA- 5360.4
GLP2 fusion

Co-Culture Studies

To determine whether the hGLP-2 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant (from both strains shown above) containing secreted human GLP-2 is added to the growth medium of Caco-2 cells and CCD-18 Co cells. The Caco-2 cell line is a continuous cell of heterogeneous human epithelial colorectal adenocarcinoma cells. As described e.g., in Jasleen et al. (Dig Dis Sci. 2002 May; 47(5):1135-40) GLP-2 stimulates proliferation and [3H]thymidine incorporation in Caco-2 and T84 cells. Additionally, GLP-2 stimulates VEGFA secretion in these cells (see., e.g., Bulut et al, Eur J Pharmacol. 2008 Jan. 14; 578(2-3):279-85.

Functional activity of bacterially secreted GLP-2 is therefore assessed by its ability to induce proliferation and VEGF secretion.

Caco-2 are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Caco-2 cells (1e6/24 well) are serum starved overnight. Titrations of either recombinant human GLP-2 (50 and 250 nM) diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for a defined time.

For cell proliferation assays, cells are harvested and resuspended in lysis buffer. The cells are assayed after 12, 24, 48, and 72 hours of incubation. Cell proliferation is measured using a Cell proliferation assay kit according to manufacturer's instruction (e.g., a Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay).

For the measurements of VEFA secretion, cells are harvested and resuspended in lysis buffer, and concentrations of GLP-2 in the medium are determined ELISA

PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity.

Competition Studies

As an additional control for specificity, a competition assay is performed. Titrations of anti-GLP-2 antibody are pre-incubated with constant concentrations of either recombinant GLP-2 or supernatants from the engineered bacteria for 15 min. Next, the supernatants/ rhIL2 solutions are added to serum-starved Cac-2 cells (1e6/well) and cells are incubated for 30 min followed by VEGFA ELISA as described above.

Example 49

Bacterial Secretion of IL-22

To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant from two engineered strains comprising IL-22 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing IL-22 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing IL-22, further comprising a deletion in degP (Table 75).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4.0 then placed on ice until further processing.

The concentration of IL-22 in the cell-free medium and in the bacterial cell extract was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL-22), according to manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Standard curves were generated using recombinant IL-22. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 75 summarizes levels of IL-22 measured in the supernatant. The data show that both hIL-22 are secreted at various levels from the different bacterial strains.

TABLE 75
IL-22 Secretion
                      IL-22
                      Production/Secretion
                      Dilution Corrected
Genotype              (ng/ml)
WT                    20.7
Lpp (delta lpp::CmR   87.6
expressing PhoA-IL22 from
Ptet)
nlpI (delta nlpI::CmR 105.4
expressing PhoA-IL22 from
Ptet)
tolA (delta tolA::CmR 623.2
expressing PhoA-IL22 from
Ptet)
PAL (delta pal::CmR   328.8
expressing PhoA-IL22 from
Ptet)

Example 50

Bacterial Secretion of IL-22 and Functional Assays

Generation of Bacterial Supernatant and Measurement of IL-22 Concentration

To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant was measured.

E. coli Nissle comprising a tet-inducible integrated construct (delta pal::CmR expressing PhoA-IL22 from Ptet) was grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-22. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the supernatant was centrifuged, and filtered through a 0.22-micron filter to remove any remaining bacteria.

The concentration of hIL-22 in the cell-free medium was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL-22), per manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Additionally, samples were diluted to ensure absence of matrix effects and to demonstrate linearity. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. The engineered bacteria comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag were determined to be secreting at 199 ng/ml supernatant.

Co-Culture Studies

To determine whether the hIL-22 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation was conducted, in which the bacterial supernatant containing secreted human IL-22 was added to the growth medium of a mammalian colonic epithelial cell line. IL-22 is known to induce the phosphorylation of STAT1 and STAT3 in Colo205 cells (see, e.g., Nagalakshmi et al., Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells. Int Immunopharmacol. 2004 May; 4(5):679-91). Functional activity of bacterially secreted IL-22 was therefore assessed by its ability to phosphorylate STAT3 in Colo205 cells.

Colo205 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Colo205 (1e6/24 well) were serum starved overnight. Titrations of either recombinant human IL-22 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria were added to cells for 30 minutes. Cells were harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) was run in triplicate for all samples, according to manufacturer's instructions. PBS-treated cells and PBS were added as negative controls. Dilutions of samples were included to demonstrate linearity. No signal was observed for wild type Nissle. Results for the engineered strain comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag are shown in FIG. 26A, and demonstrate that hIL-22 secreted from the engineered bacteria is functionally active.

Competition Studies

As an additional control for specificity, a competition assay was performed. Titrations of anti-IL22 antibody (MAB7821, R&D Systems) were pre-incubated with constant concentrations of either rhIL22 (data not shown) or supernatants from the engineered bacteria for 15 min. Next, the supernatants/ rhIL2 solutions were added to serum-starved Colo205 cells (1e6/well) and cells were incubated for 30 min followed by pSTAT3 ELISA as described above. As shown in FIG. 26B, the phospho-Stat3 signal induced by the secreted hIL-22 is competed by the hIL-22 antibody MAB7821.

Example 51

Bacterial Secretion of GLP-1

The concentration of secreted GLP-lin the bacterial supernatant from four engineered strains comprising GLP-1 constructs/strains with different ribosome binding site (RBS) strength and two different secretion tags (PhoA or OmpF) were measured and compared.

All of the constructs were tested in a deltaLpp background. Strains are described in Table 76 (and shown in FIG. 24 and FIG. 25). 20K, 100K and 67K are numbers indicating the strengths of the RBS as determined by bioinformatics on an arbitrary scale, e.g., strength of 20K<67K<100K.

Strains were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm). Cultures were diluted to an optical density of 0.5 at which time strains were induced with ATC (100 ng/mL). After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22 micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris was spun down at 20 min at 12,000 rcf at 4.0 then placed on ice until further processing.

The concentration of GLP-lin the cell-free medium and in the bacterial cell extract was measured by Abcam kit (ab184857) according to the manufacturers protocol. Standard curves were generated using recombinant GLP-1. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Results are shown in Table 76 and FIG. 25C.

TABLE 76
GLP-1 Secretion
			ng/mL
	Strain	Genotype	GLP1
	SYN2627	Δlpp TetR-pTet-20K RBS-PhoA-Glp1	3.6
	SYN2643	Δlpp TetR-pTet-100K RBS-PhoA-Glp1	26.3
	SYN2672	Δlpp TetR-pTet-20K RBS-OmpF-Glp1	2
	SYN2673	Δlpp TetR-pTet-67K RBS-OmpF-Glp1	57.6

TABLE 77
Glp1 Secretion Sequences
Description        Sequence
pTet-20K RBS-PhoA- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT
Glp1               AGAGTTATTTTACCACTCCCTATCAGTGATAGAG
SEQ ID NO: 337     AAAAGTGAACCAAAACAGAGTCATATTTAAAGG
                   AAGGTACAAAATGAAGCAGAGCACCATCGCGCT
                   TGCCCTGCTGCCGTTGCTTTTCACGCCTGTCACCA
                   AGGCTCACGATGAATTTGAGAGACATGCAGAAG
                   GAACGTTCACATCTGATGTGTCATCATATTTGGA
                   AGGCCAAGCTGCCAAAGAATTCATCGCATGGTTG
                   GTGAAAGGCCGAGGATGA
pTet-100K RBS-     TAATTCCTAATTTTTGTTGACACTCTATCATTGAT
PhoA-Glp1          AGAGTTATTTTACCACTCCCTATCAGTGATAGAG
SEQ ID NO: 338     AAAAGTGAAATAAGTTTATCAAAATAAAAGGAG
                   GTAATATATGAAGCAGAGCACCATCGCGCTTGCC
                   CTGCTGCCGTTGCTTTTCACGCCTGTCACCAAGGC
                   TCACGATGAATTTGAGAGACATGCAGAAGGAAC
                   GTTCACATCTGATGTGTCATCATATTTGGAAGGC
                   CAAGCTGCCAAAGAATTCATCGCATGGTTGGTGA
                   AAGGCCGAGGATGA
pTet-20K RBS-OmpF- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT
Glp1               AGAGTTATTTTACCACTCCCTATCAGTGATAGAG
SEQ ID NO: 339     AAAAGTGAAGTCTTCCCGATCCTTTCCCGAGCGT
                   ACAAAATGATGAAGCGTAACATCTTAGCCGTTAT
                   TGTCCCCGCATTGCTTGTGGCCGGGACGGCTAAC
                   GCACACGATGAATTTGAGAGACATGCAGAAGGA
                   ACGTTCACATCTGATGTGTCATCATATTTGGAAG
                   GCCAAGCTGCCAAAGAATTCATCGCATGGTTGGT
                   GAAAGGCCGAGGATGA
pTet-67K RBS OmpF- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT
Glp1               AGAGTTATTTTACCACTCCCTATCAGTGATAGAG
SEQ ID NO: 340     AAAAGTGAAAAAACCGCCATCAAGAGTTAAGGA
                   GGAGAATATGATGAAGCGTAACATCTTAGCCGTT
                   ATTGTCCCCGCATTGCTTGTGGCCGGGACGGCTA
                   ACGCACACGATGAATTTGAGAGACATGCAGAAG
                   GAACGTTCACATCTGATGTGTCATCATATTTGGA
                   AGGCCAAGCTGCCAAAGAATTCATCGCATGGTTG
                   GTGAAAGGCCGAGGATGA
20K RBS            CCAAAACAGAGTCATATTTAAAGGAAGGTACAA
SEQ ID NO: 341     A
67K RBS            AAAACCGCCATCAAGAGTTAAGGAGGAGAAT
SEQ ID NO: 342
100K RBS           ATAAGTTTATCAAAATAAAAGGAGGTAATAT
SEQ ID NO: 343
PhoA               ATGAAGCAGAGCACCATCGCGCTTGCCCTGCTGC
SEQ ID NO: 344     CGTTGCTTTTCACGCCTGTCACCAAGGCT
OmpF               ATGATGAAGCGTAACATCTTAGCCGTTATTGTCC
SEQ ID NO: 345     CCGCATTGCTTGTGGCCGGGACGGCTAACGCA
Glp1               CACGATGAATTTGAGAGACATGCAGAAGGAACG
SEQ ID NO: 346     TTCACATCTGATGTGTCATCATATTTGGAAGGCC
                   AAGCTGCCAAAGAATTCATCGCATGGTTGGTGAA
                   AGGCCGAGGATGA
PhoA-Glp1          ATGAAGCAGAGCACCATCGCGCTTGCCCTGCTGC
SEQ ID NO: 347     CGTTGCTTTTCACGCCTGTCACCAAGGCTCACGAT
                   GAATTTGAGAGACATGCAGAAGGAACGTTCACAT
                   CTGATGTGTCATCATATTTGGAAGGCCAAGCTGC
                   CAAAGAATTCATCGCATGGTTGGTGAAAGGCCGA
                   GGATGA
OmpF-Glp1          ATGATGAAGCGTAACATCTTAGCCGTTATTGTCC
SEQ ID NO: 348     CCGCATTGCTTGTGGCCGGGACGGCTAACGCACA
                   CGATGAATTTGAGAGACATGCAGAAGGAACGTTC
                   ACATCTGATGTGTCATCATATTTGGAAGGCCAAG
                   CTGCCAAAGAATTCATCGCATGGTTGGTGAAAGG
                   CCGAGGATGA
Terminator         AACGATTGGTAAACCCGGTGaacgcatgagAAAGCCC
SEQ ID NO: 349     CCGGAAGATCACCTTCCGGGGGCTTTtttattgcgcGG
                   ACCAAAACGAAAAAAGACGCTCGAAAGCGTCTC
                   TTTTCTGGAATTTGGTACCGAGG

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, and SEQ ID NO: 349.

Example 52

TABLE 78
Other Sequences of interest
Wild-type clbA   caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacat
(SEQ ID NO: 350) caaacataaaatacaagctcggaatacgaatcacgctatacacattgctaacagga
                 atgagattatctaaatgaggattgatatattaattggacatactagttatttcatcaaac
                 cagtagagataacttccttcactatctcaatgaggaagaaataaaacgctatgatca
                 gtttcattttgtgagtgataaagaactctatattttaagccgtatcctgctcaaaacagc
                 actaaaaagatatcaacctgatgtctcattacaatcatggcaatttagtacgtgcaaat
                 atggcaaaccatttatagtattcctcagttggcaaaaaagattattttaacctttcccat
                 actatagatacagtagccgttgctattagttctcactgcgagcttggtgtcgatattga
                 acaaataagagatttagacaactcttatctgaatatcagtcagcattatttactccaca
                 ggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttattggaaaat
                 gtggacgctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggact
                 ggattgtattgaatttcatttaacaaataaaaaactaacttcaaaatatagaggttcacc
                 tgtttatttctctcaatggaaaatatgtaactcatttctcgcattagcctctccactcatca
                 cccctaaaataactattgagctatttcctatgcagtcccaactttatcaccacgactatc
                 agctaattcattcgtcaaatgggcagaattgaatcgccacggataatctagacacttc
                 tgagccgtcgataatattgattttcatattccgtcggtggtgtaagtatcccgcataatc
                 gtgccattcacatttag
clbA knock-out   ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaa
(SEQ ID NO: 351) gacaagtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatctt
                 aacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagc
                 tcggaatacgaatcacgctatacacattgctaacaggaatgagattatctaaatgagga
                 ttgaTGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATAC
                 TTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCG
                 GAATAGGAACTAAGGAGGATATTCATATGtcgtcaaatggg
                 cagaattgaatcgccacggataatctagacacttctgagccgtcgataatattgattttc
                 atattccgtcggtgg

Example 53

Assessment of In Vitro and In Vivo Activity of Biosafety System Containing Strain

The activity of the following strains is tested:

SYN-1001 comprises a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1002 comprises a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1003 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1004 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1005 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1006 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1007 comprises a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1008 a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1009 a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1011 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1013 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1014 comprises a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

TABLE 79
Biosafety System Constructs and Sequence Components
		SEQ ID
Description	Sequence	NO
Biosafety Plasmid	ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG	352
System	GGTTATTGTCTCATGAGCGGATACATATTTGAATGT
Component-dap	ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA
A	AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG
Biosafety Plasmid	CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC
System Vector	CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG
sequences,	CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC
comprising dapA,	TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG
Kid Toxin and	AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC
R6K minimal on,	GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC
and promoter	CCAATAATTACGATTTAAATCACAGCAAACACCAC
elements driving	GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT
expression of these	TGCTGCATAACTTGACAATTAACATCCGGCTCGTAG
components, as	GGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGAG
shown in FIG.	ATCAACAATGAAAGCAATTTTCGTACTGAAACATCT
74A	TAATCATGCTGGGGAGGGTTTCTAATGTTCACGGGA
	AGTATTGTCGCGATTGTTACTCCGATGGATGAAAAA
	GGTAATGTCTGTCGGGCTAGCTTGAAAAAACTGATT
	GATTATCATGTCGCCAGCGGTACTTCGGCGATCGTT
	TCTGTTGGCACCACTGGCGAGTCCGCTACCTTAAAT
	CATGACGAACATGCTGATGTGGTGATGATGACGCT
	GGATCTGGCTGATGGGCGCATTCCGGTAATTGCCGG
	GACCGGCGCTAACGCTACTGCGGAAGCCATTAGCC
	TGACGCAGCGCTTCAATGACAGTGGTATCGTCGGCT
	GCCTGACGGTAACCCCTTACTACAATCGTCCGTCGC
	AAGAAGGTTTGTATCAGCATTTCAAAGCCATCGCTG
	AGCATACTGACCTGCCGCAAATTCTGTATAATGTGC
	CGTCCCGTACTGGCTGCGATCTGCTCCCGGAAACGG
	TGGGCCGTCTGGCGAAAGTAAAAAATATTATCGGA
	ATCAAAGAGGCAACAGGGAACTTAACGCGTGTAAA
	CCAGATCAAAGAGCTGGTTTCAGATGATTTTGTTCT
	GCTGAGCGGCGATGATGCGAGCGCGCTGGACTTCA
	TGCAATTGGGCGGTCATGGGGTTATTTCCGTTACGG
	CTAACGTCGCAGCGCGTGATATGGCCCAGATGTGC
	AAACTGGCAGCAGAAGGGCATTTTGCCGAGGCACG
	CGTTATTAATCAGCGTCTGATGCCATTACACAACAA
	ACTATTTGTCGAACCCAATCCAATCCCGGTGAAATG
	GGCATGTAAGGAACTGGGTCTTGTGGCGACCGATA
	CGCTGCGCCTGCCAATGACACCAATCACCGACAGT
	GGCCGTGAGACGGTCAGAGCGGCGCTTAAACATGC
	CGGTTTGCTGTAAGACTTTTGTCAGGTTCCTACTGT
	GACGACTACCACCGATAGACTGGAGTGTTGCTGCG
	AAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGAA
	GTCACCACGATTGTGCTTTACACGGAGTAGTCGGCA
	GTTCCTTAAGTCAGAATAGTGGACAGGCGGCCAAG
	AACTTCGTTCATGATAGTCTCCGGAACCCGTTCGAG
	TCGTTTTCCGCCCCGTGCTTTCATATCAATTGTCCGG
	GGTTGATCGCAACGTACAACACCTGTGGTACGTATG
	CCAACACCATCCAACGACACCGCAAAGCCGGCAGT
	GCGGGCAAAATTGCCTCCGCTGGTTACGGGCACAA
	CAACAGGCAGGCGGGTCACGCGATTAAAGGCCGCC
	GGTGTGACAATCAGCACCGGCCGCGTTCCCTGCTGC
	TCATGACCTGCGGTAGGATCAAGCGAGACAAGCCA
	GATTTCCCCTCTTTCCATCTAGTATAACTATTGTTTC
	TCTAGTAACATTTATTGTACAACACGAGCCCATTTT
	TGTCAAATAAATTTTAAATTATATCAACGTTAATAA
	GACGTTGTCAATAAAATTATTTTGACAAAATTGGCC
	GGCCGGCGCGCCGATCTGAAGATCAGCAGTTCAAC
	CTGTTGATAGTACGTACTAAGCTCTCATGTTTCACG
	TACTAAGCTCTCATGTTTAACGTACTAAGCTCTCAT
	GTTTAACGAACTAAACCCTCATGGCTAACGTACTAA
	GCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCA
	CGTACTAAGCTCTCATGTTTGAACAATAAAATTAAT
	ATAAATCAGCAACTTAAATAGCCTCTAAGGTTTTAA
	GTTTTATAAGAAAAAAAAGAATATATAAGGCTTTT
	AAAGCCTTTAAGGTTTAACGGTTGTGGACAACAAG
	CCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCC
	TCTCAAAGCAATTTTGAGTGACACAGGAACACTTA
	ACGGCTGACATGGGGCGCGCCCAGCTGTCTAGGGC
	GGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGAC
	AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGA
	CTGAGCCTTTCGTTTTATTTGATGCCT
Biosafety Plasmid	ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG	353
System	GGTTATTGTCTCATGAGCGGATACATATTTGAATGT
Component-	ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA
ThyA	AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG
Bio safety Plasmid	CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC
System Vector	CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG
sequences,	CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC
comprising ThyA,	TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG
Kid Toxin and	AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC
R6K minimal on,	GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC
and promoter	CCAATAATTACGATTTAAATCACAGCAAACACCAC
elements driving	GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT
expression of these	TGCTGCATAACTTGACAATTAATCATCCGGCTCGTA
components, as	GGGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGA
shown in FIG.	GATCAACAATGAAAGCAATTTTCGTACTGAAACAT
74B	CTTAATCATGCTGGGGAGGGTTTCTAATGAAACAGT
	ATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGC
	ACACAGAAAAACGACCGTACCGGAACCGGAACGCT
	TTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCA
	AGATGGATTCCCGCTGGTGACAACTAAACGTTGCC
	ACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTC
	TTCAGGGCGACACTAACATTGCTTATCTACACGAAA
	ACAATGTCACCATCTGGGACGAATGGGCCGATGAA
	AACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG
	GCGTGCCTGGCCAACGCCAGATGGTCGTCATATTGA
	CCAGATCACTACGGTACTGAACCAGCTGAAAAACG
	ACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGA
	ACGTAGGCGAACTGGATAAAATGGCGCTGGCACCG
	TGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGC
	AAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGAC
	GTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTAC
	GCGTTATTGGTGCATATGATGGCGCAGCAGTGCGAT
	CTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGAC
	ACGCATCTGTACAGCAACCATATGGATCAAACTCAT
	CTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAA
	GTTGATTATCAAACGTAAACCCGAATCCATCTTCGA
	CTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGA
	TCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTA
	AGACTTTTGTCAGGTTCCTACTGTGACGACTACCAC
	CGATAGACTGGAGTGTTGCTGCGAAAAAACCCCGC
	CGAAGCGGGGTTTTTTGCGAGAAGTCACCACGATT
	GTGCTTTACACGGAGTAGTCGGCAGTTCCTTAAGTC
	AGAATAGTGGACAGGCGGCCAAGAACTTCGTTCAT
	GATAGTCTCCGGAACCCGTTCGAGTCGTTTTCCGCC
	CCGTGCTTTCATATCAATTGTCCGGGGTTGATCGCA
	ACGTACAACACCTGTGGTACGTATGCCAACACCATC
	CAACGACACCGCAAAGCCGGCAGTGCGGGCAAAAT
	TGCCTCCGCTGGTTACGGGCACAACAACAGGCAGG
	CGGGTCACGCGATTAAAGGCCGCCGGTGTGACAAT
	CAGCACCGGCCGCGTTCCCTGCTGCTCATGACCTGC
	GGTAGGATCAAGCGAGACAAGCCAGATTTCCCCTC
	TTTCCATCTAGTATAACTATTGTTTCTCTAGTAACAT
	TTATTGTACAACACGAGCCCATTTTTGTCAAATAAA
	TTTTAAATTATATCAACGTTAATAAGACGTTGTCAA
	TAAAATTATTTTGACAAAATTGGCCGGCCGGCGCGC
	CGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTA
	CGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTC
	ATGTTTAACGTACTAAGCTCTCATGTTTAACGAACT
	AAACCCTCATGGCTAACGTACTAAGCTCTCATGGCT
	AACGTACTAAGCTCTCATGTTTCACGTACTAAGCTC
	TCATGTTTGAACAATAAAATTAATATAAATCAGCAA
	CTTAAATAGCCTCTAAGGTTTTAAGTTTTATAAGAA
	AAAAAAGAATATATAAGGCTTTTAAAGCCTTTAAG
	GTTTAACGGTTGTGGACAACAAGCCAGGGATGTAA
	CGCACTGAGAAGCCCTTAGAGCCTCTCAAAGCAAT
	TTTGAGTGACACAGGAACACTTAACGGCTGACATG
	GGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTC
	CTACTCAGGAGAGCGTTCACCGACAAACAACAGAT
	AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG
	TTTTATTTGATGCCT
Kid toxin (reverse	TTAAGTCAGAATAGTGGACAGGCGGCCAAGAACTT	354
orientation)	CGTTCATGATAGTCTCCGGAACCCGTTCGAGTCGTT
	TTCCGCCCCGTGCTTTCATATCAATTGTCCGGGGTT
	GATCGCAACGTACAACACCTGTGGTACGTATGCCA
	ACACCATCCAACGACACCGCAAAGCCGGCAGTGCG
	GGCAAAATTGCCTCCGCTGGTTACGGGCACAACAA
	CAGGCAGGCGGGTCACGCGATTAAAGGCCGCCGGT
	GTGACAATCAGCACCGGCCGCGTTCCCTGCTGCTCA
	TGACCTGCGGTAGGATCAAGCGAGACAAGCCAGAT
	TTCCCCTCTTTCCAT
dapA	ATGTTCACGGGAAGTATTGTCGCGATTGTTACTCCG	355
	ATGGATGAAAAAGGTAATGTCTGTCGGGCTAGCTT
	GAAAAAACTGATTGATTATCATGTCGCCAGCGGTA
	CTTCGGCGATCGTTTCTGTTGGCACCACTGGCGAGT
	CCGCTACCTTAAATCATGACGAACATGCTGATGTGG
	TGATGATGACGCTGGATCTGGCTGATGGGCGCATTC
	CGGTAATTGCCGGGACCGGCGCTAACGCTACTGCG
	GAAGCCATTAGCCTGACGCAGCGCTTCAATGACAG
	TGGTATCGTCGGCTGCCTGACGGTAACCCCTTACTA
	CAATCGTCCGTCGCAAGAAGGTTTGTATCAGCATTT
	CAAAGCCATCGCTGAGCATACTGACCTGCCGCAAA
	TTCTGTATAATGTGCCGTCCCGTACTGGCTGCGATC
	TGCTCCCGGAAACGGTGGGCCGTCTGGCGAAAGTA
	AAAAATATTATCGGAATCAAAGAGGCAACAGGGAA
	CTTAACGCGTGTAAACCAGATCAAAGAGCTGGTTTC
	AGATGATTTTGTTCTGCTGAGCGGCGATGATGCGAG
	CGCGCTGGACTTCATGCAATTGGGCGGTCATGGGGT
	TATTTCCGTTACGGCTAACGTCGCAGCGCGTGATAT
	GGCCCAGATGTGCAAACTGGCAGCAGAAGGGCATT
	TTGCCGAGGCACGCGTTATTAATCAGCGTCTGATGC
	CATTACACAACAAACTATTTGTCGAACCCAATCCAA
	TCCCGGTGAAATGGGCATGTAAGGAACTGGGTCTT
	GTGGCGACCGATACGCTGCGCCTGCCAATGACACC
	AATCACCGACAGTGGCCGTGAGACGGTCAGAGCGG
	CGCTTAAACATGCCGGTTTGCTGTAA
thyA	ATGAAACAGTATTTAGAACTGATGCAAAAAGTGCT	356
	CGACGAAGGCACACAGAAAAACGACCGTACCGGA
	ACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGT
	TTTAACCTGCAAGATGGATTCCCGCTGGTGACAACT
	AAACGTTGCCACCTGCGTTCCATCATCCATGAACTG
	CTGTGGTTTCTTCAGGGCGACACTAACATTGCTTAT
	CTACACGAAAACAATGTCACCATCTGGGACGAATG
	GGCCGATGAAAACGGCGACCTCGGGCCAGTGTATG
	GTAAACAGTGGCGTGCCTGGCCAACGCCAGATGGT
	CGTCATATTGACCAGATCACTACGGTACTGAACCAG
	CTGAAAAACGACCCGGATTCGCGCCGCATTATTGTT
	TCAGCGTGGAACGTAGGCGAACTGGATAAAATGGC
	GCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGT
	GGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCG
	CTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT
	TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCA
	GCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGAC
	CGGTGGCGACACGCATCTGTACAGCAACCATATGG
	ATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTC
	CGCTGCCGAAGTTGATTATCAAACGTAAACCCGAA
	TCCATCTTCGACTACCGTTTCGAAGACTTTGAGATT
	GAAGGCTACGATCCGCATCCGGGCATTAAAGCGCC
	GGTGGCTATCTAA
Kid toxin	MERGEIWLVSLDPTAGHEQQGTRPVLIVTPAAFNRVT	357
polypeptide	RLPVVVPVTSGGNFARTAGFAVSLDGVGIRTTGVVRC
	DQPRTIDMKARGGKRLERVPETIMNEVLGRLSTILT*
dapA polypeptide	MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTS	358
	AIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVI
	AGTGANATAEAISLTQRFNDSGIVGCLTVTPYYNRPS
	QEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVG
	RLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGD
	DASALDFMQLGGHGVISVTANVAARDMAQMCKLAA
	EGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKEL
	GLVATDTLRLPMTPITDSGRETVRAALKHAGLL
ThyA polypeptide	MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRF	359
	NLQDGFPLVTTKRCHLRSIIHELLWFLQGDTNIAYLHE
	NNVTIWDEWADENGDLGPVYGKQWRAWPTPDGRHI
	DQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPC
	HAFFQFYVADGKLSCQLYQRSCDVFLGLPFNIASYAL
	LVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTH
	LQLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPG
	IKAPVAI*

TABLE 80
Chromosomally Inserted Biosafety System Constructs
		SEQ
		ID
Description	Sequence	NO
Biosafety	TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT	360
Chromosomal	CTGCTGGAACAGGTGGTGAGACTCAAGGTCATGATGGA
Construct-low	CGTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGC
copy Rep (Pi)	TAAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGC
and Kis antitoxin	GAGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAG
(as shown in	AACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTG
FIG. 74C)	AAGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTG
	CTTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTG
	CCAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTT
	TAGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACT
	CCCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAG
	CTTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCA
	CCAGAACCATAGAACCATATATCTCTAGCCTTATTGGGA
	AAAAAAATAAATTCACAACGCAATTGTTAACGGCAAGC
	TTACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAAC
	TTATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATT
	ATTTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAA
	TAGCTTATACTTTTGATAAAGATGGAAATATTGAGTACA
	AATACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAA
	ATAAAGCCATTGCTGAAATTAAAAAGAAAACAGAAATA
	TCGTTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGA
	AAAATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAA
	GATGAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTT
	ATGAATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTA
	TTTGATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGA
	GGATCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCG
	AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA
	ACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTG
	GGCCTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGAC
	TtaaagtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGG
	AGGTAACAACCATGCGAGTGTTGAAGAAACATCTTAATC
	ATGCTAAGGAGGTTTTCTAATGCATACCACCCGACTGAA
	GAGGGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGC
	ACTGCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGT
	TGGCATGGTCATTGATAATGGCCGGCTGATTGTTGAGCC
	GTACAGACGCCCGCAATATTCACTGGCTGAGCTACTGGC
	ACAGTGTGATCCGAATGCTGAAATATCAGCTGAAGAAC
	GAGAATGGCTGGATGCACCGGCGACTGGTCAGGAGGAA
	ATCTGA
Biosafety	TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT	361
Chromosomal	CTTCCGGAAGACTAGGTGAGACTCAAGGTCATGATGGAC
Construct-	GTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGCT
medium copy	AAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGCG
Rep (Pi) and Kis	AGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAGA
antitoxin (as	ACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTGA
shown in FIG.	AGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTGC
74D)	TTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTGC
	CAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTTT
	AGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACTC
	CCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAGC
	TTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCAC
	CAGAACCATAGAACCATATATCTCTAGCCTTATTGGGAA
	AAAAAATAAATTCACAACGCAATTGTTAACGGCAAGCTT
	ACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAACTT
	ATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATTAT
	TTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAATA
	GCTTATACTTTTGATAAAGATGGAAATATTGAGTACAAA
	TACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAAATA
	AAGCCATTGCTGAAATTAAAAAGAAAACAGAAATATCG
	TTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGAAAA
	ATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAAGAT
	GAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTTATG
	AATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTATTTG
	ATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGAGGA
	TCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA
	GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACG
	CTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC
	CTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGACTtaaa
	gtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGGAGG
	TAACAACCATGCGAGTGTTGAAGAAACATCTTAATCATG
	CTAAGGAGGTTTTCTAATGCATACCACCCGACTGAAGAG
	GGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGCACT
	GCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGTTGG
	CATGGTCATTGATAATGGCCGGCTGATTGTTGAGCCGTA
	CAGACGCCCGCAATATTCACTGGCTGAGCTACTGGCACA
	GTGTGATCCGAATGCTGAAATATCAGCTGAAGAACGAG
	AATGGCTGGATGCACCGGCGACTGGTCAGGAGGAAATC
	TGA
Rep (Pi)	TGAGACTCAAGGTCATGATGGACGTGAACAAAAAAACG	362
	AAAATTCGCCACCGAAACGAGCTAAATCACACCCTGGCT
	CAACTTCCTTTGCCCGCAAAGCGAGTGATGTATATGGCG
	CTTGCTCCCATTGATAGCAAAGAACCTCTTGAACGAGGG
	CGAGTTTTCAAAATTAGGGCTGAAGACCTTGCAGCGCTC
	GCCAAAATCACCCCATCGCTTGCTTATCGACAATTAAAA
	GAGGGTGGTAAATTACTTGGTGCCAGCAAAATTTCGCTA
	AGAGGGGATGATATCATTGCTTTAGCTAAAGAGCTTAAC
	CTGCTCTTTACTGCTAAAAACTCCCCTGAAGAGTTAGAC
	CTTAACATTATTGAGTGGATAGCTTATTCAAATGATGAA
	GGATACTTGTCTTTAAAATTCACCAGAACCATAGAACCA
	TATATCTCTAGCCTTATTGGGAAAAAAAATAAATTCACA
	ACGCAATTGTTAACGGCAAGCTTACGCTTAAGTAGCCAG
	TATTCATCTTCTCTTTATCAACTTATCAGGAAGCATTACT
	CTAATTTTAAGAAGAAAAATTATTTTATTATTTCCGTTGA
	TGAGTTAAAGGAAGAGTTAATAGCTTATACTTTTGATAA
	AGATGGAAATATTGAGTACAAATACCCTGACTTTCCTAT
	TTTTAAAAGGGATGTGTTAAATAAAGCCATTGCTGAAAT
	TAAAAAGAAAACAGAAATATCGTTTGTTGGCTTCACTGT
	TCATGAAAAAGAAGGAAGAAAAATTAGTAAGCTGAAGT
	TCGAATTTGTCGTTGATGAAGATGAATTTTCTGGCGATA
	AAGATGATGAAGCTTTTTTTATGAATTTATCTGAAGCTG
	ATGCAGCTTTTCTCAAGGTATTTGATGAAACCGTACCTC
	CCAAAAAAGCTAAGGGGTGA
Kis antitoxin	CATACCACCCGACTGAAGAGGGTTGGCGGCTCAGTTATG	363
	CTGACCGTCCCACCGGCACTGCTGAATGCGCTGTCTCTG
	GGCACAGATAATGAAGTTGGCATGGTCATTGATAATGGC
	CGGCTGATTGTTGAGCCGTACAGACGCCCGCAATATTCA
	CTGGCTGAGCTACTGGCACAGTGTGATCCGAATGCTGAA
	ATATCAGCTGAAGAACGAGAATGGCTGGATGCACCGGC
	GACTGGTCAGGAGGAAATCTGA
RBS (low copy)	GCTGGAACAGGTGG	364
RBS (medium	TCCGGAAGACTAGG	365
copy)

Claims

1. An engineered bacterium comprising a gene sequence or gene cassette for producing one or more aryl hydrocarbon receptor (AhR) agonist(s), wherein the gene sequence or gene cassette is operably linked to a directly or indirectly inducible promoter that is not associated with the gene sequence or gene cassette in nature.

2. The engineered bacterium of claim 1, wherein the engineered bacterium comprises gene sequence for producing indole-3-acetonitrile.

3. The engineered bacterium of claim 2, wherein the engineered bacterium comprises gene sequence encoding cyp79B2 (tryptophan N-monooxygenase).

4. The genetically engineered bacteria of claim 2 or claim 3, wherein the engineered bacterium comprises gene sequence encoding cyp71a13 (indoleacetaldoxime dehydratase).

5. The genetically engineered bacteria of any of claims 2-4, wherein the engineered bacterium comprises gene sequence encoding cyp79B3 (tryptophan N-monooxygenase).

6. The genetically engineered bacteria of claim 5, wherein the cyp79B2, cyp71a13, and cyp79B3 are from Arabidopsis thaliana.

7. The bacterium of any of claims 1-6, wherein the bacterium comprises a gene or gene cassette for producing indole-3-propionic acid.

8. The genetically engineered bacteria of claim 7, wherein the engineered bacterium comprises gene sequence encoding tryptophan ammonia lyase.

9. The genetically engineered bacyteris of claim 8, wherein the tryptophan ammonia lyase is from Rubrivivax benzoatilyticus.

10. The genetically engineered bacterium of any of claims 7-9, wherein the engineered bacterium comprises one or more gene sequences encoding indole-3-acrylate reductase.

11. The genetically engineered bacterium of claim 10, wherein the ndole-3-acrylate reductase is from Clostridum botulinum.

12. The genetically engineered bacterium of any of claims 7-11, wherein the engineered bacterium comprises gene sequence encoding Tryptophan dehydrogenase (trpDH).

13. The genetically engineered bacteria of claim 12, wherein the trpDH is from Nostoc punctiforme NIES-2108.

14. The genetically engineered bacterium of any of claims 7, claim 12 and claim 13, wherein the engineered bacterium comprises gene sequence encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase).

15. The genetically engineered bacterium of claim 14, wherein the fldA is from Clostridium sporogenes.

16. The genetically engineered bacterium of any of claims 7 and claims 12-15, wherein the bacterium comprises gene sequence(s) encoding fldB and fldC (indole-3-lactate dehydratase).

17. The genetically engineered bacterium of claim 16, wherein the fldB and fldC is from Clostridium sporogenes.

18. The genetically engineered bacterium of any of claims 7 and claims 12-17, wherein the engineered bacterium comprises gene sequences encoding fldD (indole-3-acrylyl-CoA reductase).

19. The genetically engineered bacterium of claim 18, wherein the fldD is from Clostridium sporogenes.

20. The genetically engineered bacterium of any of claims 7 and claims 12-19, wherein the engineered bacterium comprises gene sequences encoding Acul (acrylyl-CoA reductase).

21. The genetically engineered bacteria of claim 20, wherein the Acul is from Rhodobacter sphaeroides.

22. The genetically engineered bacterium of any of claims 7 and claims 12-21, wherein the engineered bacterium comprises gene sequence encoding fldH1 (3-lactate dehydrogenase 1).

23. The genetically engineered bacterium of claim 22, wherein the fldH1 is from Clostridium sporogenes.

24. The genetically engineered bacterium of any of claims 7 and claims 12-23, wherein the engineered bacterium comprises gene sequence encoding fldH2 (indole-3-lactate dehydrogenase 2).

25. The genetically engineered bacteria of claim 24, wherein the fldH2 is from Clostridium sporogenes.

26. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequences encoding trpDH, fldA, fldB, flD, and fldH1.

27. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequences encoding trpDH, fldA, fldB, flD, and fldH2.

28. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequence encoding trpDH, fldA, fldB, acuI and fldH1.

29. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequence encoding trpDH, fldA, fldB, acuI and fldH2.

30. The genetically engineered bacterium of any of claims 1-29, wherein the engineered bacterium comprises gene sequence for producing tryptamine.

31. The engineered bacteria of claim 30, wherein the engineered bacterium comprises gene sequence encoding Tryptophan decarboxylase.

32. The engineered bacterium of claim 31, wherein the Tryptophan decarboxylase is from Catharanthus roseus.

33. The engineered bacterium of any of claims 1-32, wherein the engineered bacterium comprises gene sequence for producing producing indole-3-acetaldehyde.

34. The genetically engineered bacterium of claim 33, wherein the engineered bacterium comprises gene sequence encoding aro9 (L-tryptophan aminotransferase).

35. The genetically engineered bacterium of claim 33 or claim 34, wherein the engineered bacterium comprises gene sequence encoding aspC (aspartate aminotransferase.

36. The genetically engineered bacterium of any of claims 33-35, wherein the engineered bacterium comprises gene sequence encoding taal (L-tryptophan-pyruvate aminotransferase.

37. The genetically engineered bacterium of any of claims 33-36, wherein the engineered bacterium comprises gene sequence encoding staO (L-tryptophan oxidase).

38. The genetically engineered bacterium of any of claims 33-37, wherein the engineered bacterium comprises gene sequence encoding trpDH (Tryptophan dehydrogenase).

39. The genetically engineered bacterium of any of claims 33-38, wherein the engineered bacterium comprises gene sequence encoding ipdC (Indole-3-pyruvate decarboxylase).

40. The genetically engineered bacterium of claim 33, wherein the engineered bacterium comprises gene sequence encoding tdc (Tryptophan decarboxylase).

41. The genetically engineered bacterium of claim 33 or claim 40, wherein the engineered bacterium comprises gene sequence encoding tynA (Monoamine oxidase).

42. The genetically engineered bacterium of any of claims 1-41, wherein the engineered bacterium comprises gene sequence for producing indole-3-acetic acid.

43. The genetically engineered bacterium of claim 42, wherein the bacterium comprises gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase), aspC (aspartate aminotransferase), taal (L-tryptophan-pyruvate aminotransferase), staO (L-tryptophan oxidase), trpDH (Tryptophan dehydrogenase), iad1 (Indole-3-acetaldehyde dehydrogenase), AAO1 (Indole-3-acetaldehyde oxidase), ipdC (Indole-3-pyruvate decarboxylase), ipdC (Indole-3-pyruvate decarboxylase), tdc (Tryptophan decarboxylase), tynA (Monoamine oxidase), yuc2 (indole-3-pyruvate monooxygenase), IaaM (Tryptophan 2-monooxygenase), and iaaH (Indoleacetamide hydrolase).

44. The genetically engineered bacterium of any of claims 1-43, wherein the bacterium further comprises gene sequence for producing tryptophan.

45. The genetically engineered bacterium of any of claims 1-44, wherein the bacterium further comprises gene sequence encoding one or more tryptophan transporters.

46. The genetically engineered bacterium of claim 45, wherein the tryptophan transporter is selected from mtr, aroP, and tnaB.

47. The bacterium of any of claims 1-46, wherein the bacterium further comprises gene sequence for producing kynurenine.

48. The bacterium of any of claims 1-47, wherein the bacterium further comprises a gene sequence for producing kynurenic acid.

49. The bacterium of any of claims 1-48, wherein the bacterium further comprises a gene sequence for producing an indole.

50. The genetically engineered bacterium of any of claims 1-49, wherein the bacterium further comprises gene sequence encoding a non-native metabolic or satiety effector molecule.

51. The bacterium of claim 50, wherein the metabolic or satiety effector molecule is selected from a a short-chain fatty acid, butyrate, propionate, acetate, GLP-1, IL-22, IL-10, bile salt hydrolase, n-acyl-phophatidylethanolamine (NAPE), a n-acyl-ethanolamines (NAE), a ghrelin receptor antagonist, peptide YY3-36, a cholecystokinin (CCK), CCK58, CCK33, CCK22, CCK8, a bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, a serotonin receptor agonist, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN),nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA).

52. The bacterium of claim 51, wherein the metabolic or satiety effector molecule is a short-chain fatty acid.

53. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is butyrate.

54. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is propionate.

55. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is GLP1.

56. The bacterium of any of claims 1-55, wherein the gene sequence is operably linked to a directly or indirectly inducible promoter that is induced by exogenous environmental conditions.

57. The bacterium of claim 56, wherein the promoter is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.

58. The bacterium claim 57, wherein the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.

59. The bacterium of claim 58, wherein the promoter is selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.

60. The bacterium of claim 59, wherein the promoter is a FNR-inducible promoter.

61. The bacterium of any of claims 1-57, wherein the promoter is regulated by a reactive nitrogen species (RNS).

62. The bacterium of any of claims 1-57, wherein the promoter is regulated by a reactive oxygen species (ROS).

63. The bacterium of any one of claims 1-62, wherein the gene sequence and operatively linked promoter are present on a plasmid in the bacterium.

64. The bacterium of any one of claims 1-62, wherein the gene sequence and operatively linked promoter are present on a chromosome in the bacterium.

65. The bacterium of any one of claims 1-64, wherein the bacterium is an auxotroph comprising a deletion or mutation in a gene required for cell survival and/or growth.

66. The genetically engineered bacterium of claim 65, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymidine biosynthetic pathway.

67. The bacterium of any one of claims 1-66, wherein the bacterium comprises a kill switch.

68. The bacterium of any of claims 1-67, wherein the bacterium is a non-pathogenic bacterium.

69. The bacterium of claim 68, wherein the bacterium is a probiotic or a commensal bacterium.

70. The bacterium of claim 69, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.

71. The bacterium of claim 70, wherein the bacterium is Escherichia coli strain Nissle.

72. A pharmaceutically acceptable composition comprising the bacterium of any one of claims 1-71; and a pharmaceutically acceptable carrier.

73. The pharmaceutically acceptable composition of claim 72, wherein the composition is formulated for oral or rectal administration.

74. A method of treating a metabolic disease in a subject in need thereof comprising the step of administering to the subject the composition of claim 72 or claim 73.

75. The method of claim 74, wherein the disorder of condition is selected from the group consisting of: type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.