Patent Application
20180142273
Not applicable.
This disclosure generally relates to the use of recombinant microorganisms to make various products.
Reactions that catalyze the iterative formation of carbon-carbon bonds are instrumental for many metabolic pathways, such as the biosynthesis of fatty acids, polyketides, and many other molecules with applications ranging from biofuels and green chemicals to therapeutic agents. These pathways typically start with small precursor metabolites that serve as building blocks that are subsequently condensed and modified in an iterative fashion until the desired chain length and functionality are achieved.
Most iterative carbon—carbon bond forming reactions in natural biological systems take place through a Claisen condensation mechanism in which the nucleophilic α-anion of an acyl-thioester, serving as the extender unit, attacks the electrophilic carbonyl carbon of another acyl-thioester, serving as the primer. Depending on how the nucleophilic α-anion is generated, the Claisen condensation reaction can be classified as decarboxylative or non-decarboxylative.
Many natural iterative carbon chain elongation pathways, like fatty acid and polyketide biosynthesis pathways, utilize decarboxylative Claisen condensation reactions with malonyl thioesters as extender units. Their potential products include fatty acids, alcohols, polyketides, esters, alkanes and alkenes with diverse chain lengths, structures and functionalities due to usage of functionalized primers, usage of α-functionalized malonyl thioesters as extender units and diverse pathways for termination of carbon chain elongation and subsequent product modification. However, despite the structural and functional diversity of these products, the use of malonyl thioester as a C2 extender unit requires the ATP-dependent activation of acetyl-CoA to malonyl-CoA, which in turn limits the energy efficiency of these pathways. Furthermore, owing to the decarboxylation mechanism, the β-site of extender units of the decarboxylative Claisen condensation must be a carboxylic group, restricting the range of extender units and potentially limiting the diversity of products that can be generated through these carbon chain elongation pathways.
In order to overcome this limitation, we have recently implemented a novel approach by driving beta-oxidation in reverse to make fatty acids instead of degrading them (see US20130316413, WO2013036812, each incorporated by reference in its entirety for all purposes). Unlike the fatty acid biosynthesis pathway, the reversal of the β-oxidation cycle operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA). In these pathways, thiolases catalyze the non-decarboxylative Claisen condensation in which acetyl-CoA, instead of malonyl thioesters, serves as the extender unit, and subsequent β-reduction reactions by hydroxyacyl-CoA dehydrogenases (HACDs), enoyl-CoA hydratases (ECHs) and enoyl-CoA reductases (ECRs) enable iteration. Compared to pathways utilizing decarboxylative Claisen condensation, these pathways are more energy efficient due to less ATP consumption for the supply of extender unit acetyl-CoA than malonyl thioesters. However, these thiolases only utilize acetyl-CoA as the extender unit, thus limiting the functionality of synthesized products. A novel non-decarboxylative Claisen condensation reaction able to accept wider range of extender units and proceed in an iterative manner is required to diversify the product range of carbon-chain elongation.
This disclosure demonstrates a general CoA-dependent carbon elongation platform based on the use of de novo thiolase-catalyzed non-decarboxylative Claisen condensation which accepts functionalized primers and extender units, along with suitable HACDs, ECHs and ECRs (
This disclosure generally relates to the use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing an iterative carbon chain elongation pathway that uses functionalized extender units. The core enzymes in the pathway include thiolases, dehydrogenases, dehydratases and reductases. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized β-keto acyl-CoA. Dehydrogenases convert alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratases convert alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductases convert alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and either acetyl-CoA or the aforementioned alpha-functionalized extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different β-reduction degrees.
This disclosure demonstrates a general CoA-dependent carbon elongation platform based on the use of thiolase-catalyzed non-decarboxylative Claisen condensations that accept alpha-functionalized extender units, along with suitable hydroxyacyl-CoA dehydrogenases (HACDs), enoyl-CoA hydratases (ECHs) and enoyl-CoA reductases (ECRs). A wide-range of alpha-functionalized product families (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives) can be obtained through this iterative platform.
The technology entails developing a new pathway that is based on native or engineered thiolases capable of catalyzing the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit. This has been reported in neither the scientific, peer-reviewed literature nor the patent literature.
The process involves performing traditional fermentations using industrial organisms (such as E. coli, S. cerevisiae) that convert different feedstocks into longer-chain products (e.g. alpha-functionalized carboxylic acids, alcohols, amines, and their beta-, and omega-functionalized derivatives or hydrocarbons). These organisms are considered workhorses of modern biotechnology. Media preparation, sterilization, inoculum preparation, and fermentation are the main steps of the process.
As used herein, a “primer” is a starting molecule for iterative carbon elongation platform. The “initial primer” or “initiating primer” can be simply acetyl-CoA or other unsubstituted or functionalized acyl-CoAs. As the chain grows by adding extender units in each cycle, the primer will accordingly increase in size.
As used herein, an “extender unit” is the donor of carbons in each cycle of the iterative carbon elongation platform. In this disclosure, the extender unit is alpha-functionalized acetyl-CoAs.
Thiolases are ubiquitous enzymes that have key roles in many vital biochemical pathways, including the beta-oxidation pathway of fatty acid degradation and various biosynthetic pathways. Members of the thiolase family can be divided into two broad categories: degradative thiolases (EC 2.3.1.16), and biosynthetic thiolases (EC 2.3.1.9). The forward and reverse reactions are shown below:
These two different types of thiolase are found both in eukaryotes and in prokaryotes: acetoacetyl-CoA thiolase (EC:2.3.1.9) and 3-ketoacyl-CoA thiolase (EC:2.3.1.16). 3-ketoacyl-CoA thiolase (also called thiolase I) has a broad chain-length specificity for its substrates and is involved in degradative pathways such as fatty acid beta-oxidation. Acetoacetyl-CoA thiolase (also called thiolase II) is specific for the thiolysis of acetoacetyl-CoA and involved in biosynthetic pathways such as poly beta-hydroxybutyric acid synthesis or steroid biogenesis.
Furthermore, the degradative thiolases can be made to run in the forward direction by building up the level of left hand side reactants (primer and extender unit), thus driving the equilibrium in the forward direction and/or by overexpressing same or by expressing a mutant of same.
As used herein, a “thiolase” is an enzyme that catalyzes the condensation of an unsubstituted or functionalized acyl-CoA thioester with alpha-functionalized acetyl-CoA as the carbon donor for chain elongation to produce an unsubstituted or omega-functionalized alpha-functionalized β-keto acyl-CoA in a non-decarboxylative condensation reaction:
As used herein, a “hydroxyacyl-CoA dehydrogenase” or “HACD”, is an enzyme that catalyzes the reduction of an unsubstituted or omega-functionalized alpha-functionalized β-keto acyl-CoA to an unsubstituted or omega-functionalized alpha-functionalized β-hydroxy acyl-CoA:
As used herein, an “enoyl-CoA hydratase” or “ECH” is an enzyme that catalyzes the dehydration of an unsubstituted or omega-functionalized or alpha-functionalized β-hydroxy acyl-CoA to an unsubstituted or omega-functionalized or alpha-functionalized enoyl-CoA:
As used herein, an “enoyl-CoA reductase” or “ECR” is an enzyme that catalyzes the reduction of an unsubstituted or omega-functionalized or alpha-functionalized transenoyl-CoA to an unsubstituted or omega-functionalized of alpha-functionalized acyl-CoA:
As used herein, “termination pathway” refers to one or more enzymes (or genes encoding same) that will pull reaction CoA thioester intermediates out the iterative cycle and produce the desired end product.
As used herein, an “alpha functionalized product” is a carboxylic acid, alcohols, hydrocarbons, or amine, wherein the alpha position is the second carbon and has an R group that is not hydrogen (R preferably being e.g., alkyl, aryl, —OH, —COOH, or —X, but including others). Note that the second carbon is defined with respect to the—coA end, and the numbering is retained even when the—coA is removed. Such alpha functionalized products can be further modified herein, and thus include beta-, and omega-functionalized derivatives.
As used herein, the expressions “microorganism,” “microbe,” “strain” and the like may be used interchangeably and all such designations include their progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
As used herein, reference to a “cell” is generally understood to include a culture of such cells, as the work described herein is done in cultures having 109-15 cells.
As used herein, “growing” cells used it its art accepted manner, referring to exponential growth of a culture of cells, not the few cells that may not have completed their cell cycle at stationary phase or have not yet died in the death phase or after harvesting.
As used in the claims, “homolog” means an enzyme with at least 50% identity to one of the listed sequences and also having the same general catalytic activity, although of course Km, Kcat and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).
Reference to proteins herein can be understood to include reference to the gene encoding such protein. Thus, a claimed “permease” protein can include the related gene encoding that permease. However, it is preferred herein to refer to the protein by standard name per ecoliwiki or HUGO since both enzymatic and gene names have varied widely, especially in the prokaryotic arts.
Once an exemplary protein is obtained, many additional examples of proteins with similar activity can be identified by BLAST search. Further, every protein record is linked to a gene record, making it easy to design overexpression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them. But, if necessary, new clones can be prepared based on available sequence information using RT-PCR techniques. Thus, it should be easily possible to obtain all of the needed enzymes for overexpression.
Another way of finding suitable enzymes/proteins for use in the invention is to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme. An enzyme that thus be obtained, e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein. In addition, many sites provide lists of proteins that all catalyze the same reaction.
Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides that encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be “optimized” for expression in E. coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.
Initial cloning experiments have proceeded in E. coli for convenience since most of the required genes are already available in plasmids suitable for bacterial expression, but the addition of genes to bacteria is of nearly universal applicability. Indeed, since recombinant methods were invented in the 70's and are now so commonplace, even school children perform genetic engineering experiments using bacteria. Such species include e.g., Bacillus, Streptomyces, Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus, or any of the completely sequenced bacterial species. Indeed, hundreds of bacterial genomes have been completely sequenced, and this information greatly simplifies both the generation of vectors encoding the needed genes, as well as the planning of a recombinant engineering protocol. Such species are listed along with links at http://en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes.
Additionally, yeasts, such as Saccharomyces, are a common species used for microbial manufacturing, and many species can be successfully transformed. Indeed, yeast are already available that express recombinant thioesterases—one of the termination enzymes described herein—and the reverse beta oxidation pathway has also been achieved in yeast. Other species include but are not limited to Candida, Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica, to name a few.
It is also possible to genetically modify many species of algae, including e.g., Spirulina, Apergillus, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica, and the like. Indeed, the microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
Furthermore, a number of databases include vector information and/or a repository of vectors and can be used to choose vectors suitable for the chosen host species. See e.g., AddGene.org which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See also Plasmid Information Database (PlasmID) and DNASU having over 191,000 plasmids. A collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.
The enzymes can be added to the genome or via expression vectors, as desired. Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via plasmid or other vector. Initial experiments may employ expression plasmids hosting 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for long term stability.
Still further improvements in yield can be had by reducing competing pathways, such as those pathways for making e.g., acetate, formate, ethanol, and lactate, and it is already well known in the art how to reduce or knockout these pathways. See e.g., the Rice patent portfolio by Ka-Yiu San and George Bennett (U.S. Pat. No. 7,569,380, U.S. Pat. No. 7,262,046, U.S. Pat. No. 8,962,272, U.S. Pat. No. 8,795,991) and patents by these inventors (U.S. Pat. No. 8,129,157 and U.S. Pat. No. 8,691,552) (each incorporated by reference herein in its entirety for all purposes). Many others have worked in this area as well.
In calculating “% identity” the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity=number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova T A & Madden T L (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI web site. The default parameters were used, except the filters were turned OFF.
“Operably associated” or “operably linked”, as used herein, refer to functionally coupled nucleic acid or amino acid sequences.
“Recombinant” is relating to, derived from, or containing genetically engineered material. In other words, the genetics of an organism was intentionally manipulated by the hand of man in some way.
“Reduced activity” is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species (e.g., the wild type gene in the same host species). Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codons, by frame shift mutation, and the like. All reduced activity genes or proteins are signified herein by “−”.
By “null” or “knockout” what is meant is that the mutation produces undetectable active protein. A gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can also completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All null mutants herein are signified by Δ.
“Overexpression” or “overexpressed” is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any detectable expression in a species that lacks the activity altogether. Preferably, the activity is increased 100-500% or even ten fold. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. All overexpressed genes or proteins are signified herein by “+”.
In certain species it is possible to genetically engineer the endogenous protein to be overexpressed by changing the regulatory sequences or removing repressors. However, overexpressing the gene by inclusion on selectable plasmids or other vectors that exist in hundreds of copies in the cell may be preferred due to its simplicity and ease of exerting externals controls, although permanent modifications to the genome may be preferred in the long term for stability reasons.
The term “endogenous” or “native” means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated, or placed under the control of a promoter that results in overexpression or controlled expression of said gene. Thus, genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be overexpressed.
“Expression vectors” are used in accordance with the art accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expressions vectors also exist.
As used herein, “inducible” means that gene expression can be controlled by the hand of man, by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac operon, inducible by IPTG, the yeast AOX1 promoter inducible with methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.
As used herein, an “integrated sequence” means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector. It will still be expressible, and preferably is inducible as well.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.
The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.
The phrase “consisting of” is closed, and excludes all additional elements.
The phrase “consisting essentially of” excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention, such as instructions for use, buffers, background mutations that do not effect the invention, and the like.
The following abbreviations are used herein:
This disclosure generally relates to the use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing a novel iterative carbon chain elongation pathway that uses functionalized extender units to grow a carbon chain by two carbon units.
The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized β-keto acyl-CoA. Dehydrogenase converts alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratase converts alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA.
The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned omega-functionalized extender unit in subsequent turns of the cycle. Various termination pathways
(
Thioesterase or CoA transferase or phosphotransacylase+carboxylate kinase can terminate the platform by converting the alpha-functionalized acyl-CoAs to alpha-functionalized carboxylic acids. If alpha-functionalized carboxylic acids has keto group at the beta-site, it can then be converted to ketone through reactions by beta-keto acid decarboxylase. Acyl-CoA reductases can terminate the platform by converting the alpha-functionalized acyl-CoAs to alpha-functionalized aldehydes. Alpha-functionalized aldehydes can then be converted to alpha-functionalized alcohols and alpha-functionalized amines through reactions by alcohol dehydrogenase and transaminase respectively.
This disclosure also relates to a novel primary alcohol synthesis incorporating the proposed iterative platform using glycolyl-CoA (alpha-hydroxy acetyl-CoA) as the extender unit. When the platform uses glycolyl-CoA as the extender unit, it generates alpha-hydroxyacyl-CoA, which can be converted to primary alcohol by termination pathways selected from: a) 2-hydroxyacyl-CoA lyase (HACL) that converts alpha-hydroxyacyl-CoA to primary aldehyde with one less carbon and formyl-CoA, and alcohol dehydrogenase subsequently converts the primary aldehyde to primary alcohol; b) acid-forming termination enzyme selected from thioesterase, CoA transferase and phosphotransacylase+carboxylate kinase that converts alpha-hydroxyacyl-CoA to alpha-hydroxy acid, keto-dehydrogenase that converts alpha-hydroxy acid to alpha-keto acid, alpha-keto acid decarboxylase that converts alpha-keto acid to primary aldehyde with one less carbon and alcohol dehydrogenase subsequently converts the primary aldehyde to primary alcohol.
Many examples of thiolase enzymes which can potentially catalyze the non-decarboxylative condensation of an acyl-CoA primer and acetyl-CoA extender unit are provided herein and Table 1 provides several additional examples which can also serve as templates for engineered variants:
E. coli atoB
E. coli yqeF
E. coli fadA
E. coli fadI
Streptomyces collinus fadA
Ralstonia eutropha bktB
Pseudomonas sp. Strain B13 catF
E coli paaJ
Pseudomonas putida pcaF
Rhodococcus opacus pcaF
Streptomyces sp. pcaF
Ralstonia eutropha phaA
Clostridium acetobutylicum thlA
Clostridium acetobutylicum thlB
This technology takes the above thiolase initiated pathway one step further to make alpha functionalized products. The method entails developing a new pathway that is based on native or engineered thiolases capable of catalyzing the condensation of either unsubstituted or functionalized acyl-CoA primers with an omega-functionalized acetyl-CoA as the extender unit. This has been reported in neither the scientific, peer-reviewed literature nor the patent literature.
Materials that can be used with the invention include those in Tables 2-5 below.
E. coli PaaK E. coli sucCD E. coli fadK E. coli fadD E. coli prpE
E. coli menE
Penicillium
chrysogenum phl
Salmonella
typhimurium LT2 prpE
Bacillius subtilis bioW
Cupriavidus
basilensis hmfD
Rhodopseudomonas
palustris badA
R. palustris hbaA
Pseudomonas
aeruginosa PAO1
Arabidopsis thaliana
E. coli atoD
E. coli atoA
E. coli scpC
Clostridium kluyveri
Clostridium
kluyveri
Clostridium
acetobutylicum ctfAB
Pseudomonas putida
Megasphaera elsdenii
Acidaminococcus
fermentans gctAB
Acetobacter aceti
E. coli ydiF
Clostridium
acetobutylicum ptb
Enterococcus faecalis
Salmonella
enterica
Clostridium
acetobutylicum buk
Enterococcus faecalis
Salmonella
enterica
E. coli atoB E. coli yqeF E. coli fadA E. coli fadI Ralstonia eutropha bktB Pseudomonas sp. Strain B13 catF E coli paaJ Pseudomonasputida pcaF Rhodococcusopacus pcaF
Streptomyces sp.
Ralstonia eutropha
Clostridium
acetobutylicum thlA
Clostridium
acetobutylicum thlB
Pseudomonas
putida fadA
P. putida fadAx
Acinetobacter sp.
E. coli paaJ
E. coli fadB E. coli fadJ E. coli paaH P. putida fadB P. putida fadB2x Acinetobacter sp. ADP1 dcaH Ralstoniaeutrophus phaB
Clostridium
acetbutylicum hbd
E. coli fabG
E. coli fadB E. coli fadJ E. coli paaF P. putida fadB P. putida fadB1x Aceinetobacter sp. ADP1 dcaE Clostridiumacetobutylicum crt
Aeromonas caviae
E. coli fabA
E. coli fabZ
Euglena gracillis TER Treponemadenticola TER Clostridiumacetobutylicum TER E. coli fabI
Enterococcus
faecalis fabK
bacillus subtilis
Vibrio cholerae
E. coli fadE
E. coli ydiO
E. coli tesA E. coli tesB E. coli yciA E. coli fadM E. coli ydiI E. coli ybgC
E. coli paal
Mus musculus
Lycopersicon
hirsutum f
glabratum mks2
Alcanivorax
borkumensis tesB2
Fibrobacter
succinogenes
Prevotella
ruminicola Pr655
Prevotella
ruminicola Pr1687
E. coli atoD
E. coli atoA
E. coli scpC
Clostridium kluyveri
Clostridium kluyveri
Clostridium
acetobutylicum
Pseudomonas
putida pcalJ
Megasphaera
elsdenii pct
Acidaminococcus
fermentans gctAB
Acetobacter aceti
E. coli ydiF
Clostridium
acetobutylicum ptb
Enterococcus
faecalis ptb
Salmonella
enterica
Clostridium
acetobutylicum buk
Enterococcus
faecalis buk
Salmonella
enterica
Acinetobacter
calcoaceticus acr1 Acinetobacter sp Strain M-1 acrM Clostridiumbeijerinckii ald
E. coli eutE
Salmonella
enterica
Marinobacter
aquaeolei VT8
E. coli mhpF
Clostridium kluyveri
E. coli betA E. coli dkgA E. coli eutG E. coli fucO E. coli ucpA E. coli yahK
E. coli ybbO
E. coli ybdH
E. coli yiaY
E. coli yjgB
Marinobacter
aquaeolei VT8
Saccharomyces
cerevisiae
Clostridium kluyveri
Acinetobacter sp.
Arbidopsis
thaliana At3g22200 Alcaligenesdenitrificans AptA Bordetellabronchiseptica
Bordetella
parapertussis
brucella menlitensis
Burkholderia
pseudomallei
Chromobacterium
violaceum CV2025
Oceanicola
granulosus
Paracoccus
denitrificans
Pseudogulbenkiania
ferrooxidans ω-
Pseudomonas
putida ω-TA
Ralstonia
solanacearum ω-
Rhizobium meliloti
Vibrio fluvialis ω-
Mus musculus
Flavobacterium
lutescens lat
Streptomyces
clavuligerus lat
E. coli gabT
E. coli puuE
E. coli ygjG
Lycopersicon
hirsutum f glabratum mks1 Clostridiumacetobutylicum adc
Clostridium
beijerinckii adh E. coli serA Gordonia sp. TY-5 adh1 Gordonia sp. TY-5 adh2
Gordonia sp. TY-5
Rhodoccoccus ruber
Acidaminococcus
fermentans hgdH
E. coli ldhA
E. coli lldD
E. coli leuB
Lactococcus lactis kivd Saccharomycescerevisiae PDC1 S. cerevisiae PDC5 S. cerevisiae PDC6 S. cerevisiae
S. cerevisiae THI3
Zymomonas
mobilis pdc
E. coli betA E. coli dkgA E. coli eutG E. coli fucO E. coli ucpA E. coli yahK E. coli ybbO E. coli ybdH
E. coli yiaY
E. coli yjgB
Saccharomyces
cerevisiae ADH6
Clostridium kluyveri
Acinetobacter sp.
Homo sapiens hacl1 Rattus norvegicus hacl1 Dictyosteliumdiscoideum hacl1 Mus musculus hacl1
All strains used in this study are listed in Table 6. Gene deletions were performed using P1 phage transduction with single-gene knockout mutants from the National BioResource Project (NIG, Japan) as the specific deletion donor. The λDE3 prophage, carrying the T7 RNA polymerase gene and lacIq, was integrated into the chromosome through λDE3 lysogenization kit (Novagen, Darmstadt, Germany). All strains were stored in 32.5% glycerol stocks at −80° C. Plates were prepared using LB medium containing 1.5% agar, and appropriate antibiotics were included at the following concentrations: ampicillin (100 μg/mL), spectinomycin (50 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (34 μg/mL).
All plasmids used in this study and oligonucleotides used in their construction are listed in Tables 6 and 7. Plasmid based gene overexpression was achieved by cloning the desired gene(s) into either pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes using In-Fusion PCR cloning technology (Clontech Laboratories, Inc., Mountain View, Calif.). Cloning inserts were created via PCR of ORFs of interest from their respective genomic or codon-optimized DNA with Phusion polymerase (Thermo Scientific, Waltham, Mass.). E. coli genes were obtained from genomic DNA, while heterologous genes were synthesized by GenScript (Piscataway, N.J.) or GeneArt (Life Technologies, Carlsbad, Calif.) with codon optimization except for bktB, phaB1, and pct, which were amplified from genomic DNA or cDNA of their source organisms. The resulting In-Fusion products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, Calif.) and PCR identified clones were confirmed by DNA sequencing.
E. coli Strains
S. cerevisiae strains
Fermentation medium and conditions: The minimal medium designed by Neidhardt et al. with 125 mM MOPS and Na2HPO4 in place of K2HPO4 (1.48 mM for fermentations in flasks; 2.8 mM for fermentations in bioreactors), supplemented with 20 g/L glycerol, 10 g/L tryptone, 5 g/L yeast extract, 100 μM FeSO4, 5 mM calcium pantothenate, 5 mM (NH4)2SO4, and 30 mM NH4Cl was used for all fermentations unless otherwise stated. Neutralized 20 mM glycolic acid or propionic acid was supplemented as needed. Antibiotics (50 mg/mL carbenicillin and 50 mg/mL spectinomycin) were included when appropriate. All chemicals were obtained from Fisher Scientific Co. (Pittsburg, Pa.) and Sigma-Aldrich Co. (St. Louis, Mo.).
Unless otherwise stated, fermentations were performed in 25 mL Pyrex Erlenmeyer flasks (narrow mouth/heavy duty rim, Corning Inc., Corning, N.Y.) filled with 20 mL fermentation medium and sealed with foam plugs filling the necks. A single colony of the desired strain was cultivated overnight (14-16 h) in LB medium with appropriate antibiotics and used as the inoculum (1%). After inoculation, flasks were incubated in a NBS 124 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, N.J.) at 200 rpm and 37° C., except fermentations supplemented with phenylacetic acid or isobutyric acid in which the temperature was 30° C. When optical density (550 nm, OD550) reached ˜0.3-0.5, 5 μM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added for plasmid based gene expression in all cases except the following: 1 μM IPTG was used for adipic acid production from glycerol without succinic acid supplementation and 10 μM IPTG was used during production of ω-phenylalkanoic acids. For induction of controlled chromosomal expression constructs, 0.1 mM cumate and 15 ng/mL anhydrotetracycline were also added when appropriate. Flasks were then incubated under the same conditions for 48 h post-induction unless otherwise stated.
Additional fermentations were conducted in a SixFors multi-fermentation system (Infors HT, Bottmingen, Switzerland) with an air flow rate of 2 N L/hr, independent control of temperature (37° C.), pH (controlled at 7.0 with NaOH and H2SO4), and stirrer speed (720 rpm). Tiglic acid fermentations used the previously described fermentation media with 30 g/L glycerol, the inclusion of 5 μM sodium selenite, and 5 μM IPTG. Propionic acid (20 mM) was added at 0, 24, and 48 h. Pre-cultures were grown in 25 mL flasks as described above, incubated for 4 h post-induction, and used for inoculation as described above.
Fermentations with glycolyl-CoA as a primer were conducted in 250 mL Erlenmeyer Flasks filled with 50 mL LB media supplemented with 10 g/L glucose and appropriate antibiotics. The cultivation of inoculum was same as above but 2% inoculation was used. After inoculation, cells were cultivated at 30° C. and 250 rpm in a NBS 124 Benchtop Incubator Shaker until an optical density of ˜0.8 was reached, at which point IPTG (0.1 mM) and neutralized glycolic acid (40 mM) were added. Flasks were then incubated under the same conditions for 96 h post induction.
GC sample preparation: Sample preparation was conducted as follows: 2 mL culture supernatant samples were transferred to 5 mL glass vials (Fisher Scientific Co., Fair Lawn, N.J., USA) and 80 μL of 50% H2SO4 and 340 μL of 30% NaCl solution were added for pH and ionic strength adjustment, respectively. Tridecanoic acid (final concentration 50 mg/L) was added as internal standard and 2 mL of hexane-MTBE (1:1) added for extraction. The bottles were sealed with Teflonlined septa (Fisher Scientific Co., Fair Lawn, N.J., USA), secured with caps, and rotated at 60 rpm for 120 min. The samples were then centrifuged for 2 min at 2,375×g to separate the aqueous and organic layers. 1 mL of the dry organic layer was transferred into a 2 mL borosilicate glass vial, dried under N2, and re-suspended in 100 μL of pyridine. After vortexing, 100 μL of BSTFA (N, O-bis(trimethylsilyl)trifluoroacetamide) was added, the samples were heated at 70° C. for 30 min, dried under N2 and re-suspended in 1 mL hexane for analysis.
GC-MS metabolite identification: Except identifications of 2,3-dihydroxybutyric acid, metabolite identification was conducted via GC-MS in an Agilent 7890A GC system (Agilent Technologies, Santa Clara, Calif.), equipped with a 5975C inert XL mass selective detector (Agilent) and Rxi-5Sil column (0.25 mm internal diameter, 0.10 mm film thickness, 30 m length; Restek, Bellefonte, Pa.). The sample injection amount was 2 mL with 40:1 split ratio. The injector and detector were maintained at 280° C. The column temperature was held initially at 35° C. for 1 min and increased to 200° C. at the rate of 6° C./min, then to 270° C. at the rate of 30° C./min. That final temperature was maintained for 1 min before cooling back to initial temperature. The carrier gas was helium (2.6 mL/min, Matheson Tri-Gas, Longmont, Colo.).
Identification of 2,3-dihydroxybyturic acid was conducted by the Baylor College of Medicine Analyte Center (bcm.edu/research/centers/analyte, Houston, Tex.). An Agilent 6890 GC system (Agilent Technologies, Santa Clara, Calif.), equipped with a 5973 mass selective detector (Agilent Technologies) and HP-5ms column (Agilent Technologies) was used. Sample extraction was conducted using Agilent Chem Elut liquid extraction columns (Agilent Technologies) according to manufacturer protocols.
HPLC metabolite quantification: The concentration of products were determined via ion-exclusion HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, Md.) equipped with an HPX-87H organic acid column (Bio-Rad, Hercules, Calif.) with operating conditions to optimize peak separation (0.3 ml/min flow rate, 30 mM H2SO4 mobile phase, column temperature 42° C.).
In vitro enzyme assay: Purified HACL1 was tested for its native catabolic activity by assessing its ability to cleave 2-hydroxyhexadecanoyl-CoA to pentadecanal and formyl-CoA. Enzyme assays were performed in 50 mM tris-HCl pH 7.5, 0.8 mM MgCl2, 0.02 mM TPP, 6.6 μM BSA, and 0.3 mM 2-hydroxyhexadecanoyl-CoA. The assay mixtures were incubated for one hour at 37° C., after which the presence of pentadecanal was assessed by extraction with hexane and analysis by GC-FID.
2-hydroxyhexadecanoyl-CoA was prepared by the n-hydroxysuccinimide method. In summary, the n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid is prepared by reacting n-hydroxysuccinimide with the acid in the presence of dicyclohexylcarbodiimide. The product was filtered and purified by recrystallization from methanol to give pure n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid. The ester was reacted with CoA-SH in presence of thioglycolic acid to give 2-hydroxyhexadecanoyl-CoA. The 2-hydroxyhexadecanoyl-CoA was purified precipitation using perchloric acid, filtration, and washing the filtrate with perchloric acid, diethyl ether, and acetone.
For specific activity assays (reported in μmol substrate/mg protein/min) these supernatant fractions were utilized and protein concentration was established using the Bradford Reagent (Thermo Sci.) using BSA as the protein standard.
Enzyme purification: A plasmid containing the codon optimized gene encoding human HIS-tagged HACL1 was constructed as described. The resulting construct was transformed into S. cerevisiae InvSC1 (Life Tech.). The resulting strain was cultured in 50 mL of SC-URA media containing 2% glucose at 30° C. for 24 hours. The cells were pelleted and the required amount of cells were used to inoculate a 250 mL culture volume of SC-URA media containing 0.2% galactose, 1 mM MgCl2, and 0.1 mM thiamine to 0.4 OD600. After 20 hours incubation with shaking at 30° C., the cells were pelleted and saved.
When needed, the cell pellets were resuspended to an OD600 of approximately 100 in a buffer containing 50 mM potassium phosphate pH 7.4, 0.1 mM thiamine pyrophosphate, 1 mM MgCl2, 0.5 mM AEBSF, 10 mM imidazole, and 250 units of Benzonase nuclease. To the cell suspension, approximately equal volumes of 425-600 μm glass beads were added. Cells were broken in four cycles of 30 seconds of vortexing at 3000 rpm followed by 30 seconds on ice. The glass beads and cell debris were pelleted by centrifugation and supernatant containing the cell extract was collected. The HIS-tagged HACL1 was purified from the cell extract using Talon Metal Affinity Resin as described above, with the only modification being the resin bed volume and all subsequent washes were halved. The eluate was collected in two 500 μL fractions.
Expression and purification of the desired protein can be confirmed by running cell pellet sample and eluate on SDS-PAGE.
We demonstrated several cases of the iterative system can synthesize alpha-functionalized small molecules through the use of alpha-functionalized forms of acetyl-CoA as the extender unit. One case used of propionyl-CoA as the extender unit. To implement this, P. putida FadAx (thiolase), FadB2x (HACD), FadB1x (ECH), and E. coli FabI (ECR) were used with Pct for activation of exogenous propionic acid. Expression in JC01(DE3) resulted in the production of 2-methylbutyric acid (75 mg/L) and tiglic acid (573 mg/L) (
Interestingly, 2-methylpentanoic acid (49 mg/L) and (E)-2-methyl-2-pentenoic acid (84 mg/L) were also synthesized, as the result of propionyl-CoA serving as both the primer and the extender unit. Products resulting from non-functionalized extender units (acetyl-CoA) with acetyl-CoA or propionyl-CoA priming were also observed, demonstrating the nonspecific activity of the thiolase (and subsequent β-reduction enzymes). This represents a potential area for further improvement through the selection and engineering of a thiolase with maximal specificity for the desired condensation. Additional alpha-functionalization was demonstrated with glycolyl-CoA (i.e. α-hydroxylated acetyl-CoA) as the extender unit, which with acetyl-CoA priming supported the synthesis of 2,3-dihydroxybutyric acid (
The ability of the alpha-functionalization system to support high product titers was investigated by improving tiglic acid production. Omission of ECR and manipulation of the termination pathway through deletion of native thioesterases and controlled overexpression of YdiI, a thioesterase previously shown to act effectively on α, β-unsaturated enoyl-CoAs, resulted in further improvement, from 573 mg/L to 1.39 g/L (
The host strains and plasmids used for production of above products are summarized in Table 8.
We also successfully expressed Homo sapiens 2-hydroxyacyl-CoA lyase HACL1 in Saccharomyces cerevisiae and Escherichia coli (
We believe that, pathway and process optimization, in line with industrial biotechnology approaches, can further improve performance for a specific target product, as the underlying carbon and energy efficiency enables the feasibility of further advancing product titer, rate, and yield. Important areas include generating and balancing pools of priming and extender units and optimization of required pathway enzymes for a given target product. The former can exploit previously developed pathways for primers and extender units, whereas the latter includes identifying and engineering enzymes that may be flux limiting due to suboptimal enzyme specificity or activity. These approaches will be continually aided by developments in protein and metabolic engineering and synthetic and systems biology.
The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli—B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHT01 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids pAMβ1 and pBS72 can be used. Several other suitable expression systems are available. Since the FAS genes are ubiquitous, the invention is predicted to function in Bacillus.
The above experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Several yeast E. coli shuttle vectors are available for ease of the experiments. Since the FAS genes are ubiquitous, the invention is predicted to function in yeast, especially since yeasts are already available with exogenous functional TE genes and the reverse beta oxidation pathway has also been made to run in yeast.
Each of the following is incorporated by reference herein in its entirety for all purposes:
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The following claims are provided to add additional clarity to this disclosure. Future applications claiming priority to this application may or may not include the following claims, and may include claims broader, narrower, or entirely different from the following claims. Further, any detail from any claim may be combined with any other detail from another claim, even if not yet so combined.
This application claims priority to U.S. Ser. No. 62/148,123, ITERATIVE PLATFORM FOR THE SYNTHESIS OF ALPHA FUNCTIONALIZED PRODUCTS, filed Apr. 15, 2015 and expressly incorporated by reference herein in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/27873 | 4/15/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62148123 | Apr 2015 | US |
