Patent Application
20100151544
The present invention relates to metabolic engineering issues related to flux determinism in core primary-metabolism pathways. In particular, the present invention relates to alcohol (e.g., butanol) production and selectivity, and related systems and methods thereof.
Clostridium acetobutylicum, included in the genus Clostridium, is a commercially valuable bacterium. Clostridium acetobutylicum is used to produce acetone, butanol, and ethanol from starch using the ABE process (Acetone Butanol Ethanol process) for industrial purposes such as gunpowder and Cordite (using acetone) production. The A.B.E. process was an industry standard until the late 1940s, when low oil costs drove more-efficient processes based on hydrocarbon cracking and petroleum distillation techniques. C. acetobutylicum also produces acetic acid (vinegar), butyric acid (a substance that smells like vomit), carbon dioxide, and hydrogen. Improved methods for producing butanol from Clostridium acetobutylicum are needed.
Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (e.g., butanol, acetone and ethanol) production and solvent tolerance, thus demonstrating that, for example, further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies.
Experiments conducted during the course of developing embodiments of the present invention demonstrated that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) resulted in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In addition, experiments conducted during the course of developing the present invention demonstrated that the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotranbutyrylase ptb promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to, for example, early production of high alcohol (butanol plus ethanol) titers and overall solvent titers of 30 g/L. Metabolic flux analysis revealed the depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, the impact of thiolase (thl) overexpression was examined. The combined thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation.
Accordingly, embodiments of the present invention provide improved methods for alcohol formation and selectivity yields. In some embodiments, the present invention provides, for example, systems and methods to accelerate and enhance alcohol (e.g., butanol) production and selectivity in organisms (e.g., solventogenic clostridia) by, for example, using different combinations of higher aldehyde and alcohol dehydrogenases and/or thiolase expression combined with, for example, CoA transferase downregulation and also by metabolic engineering strategies aimed at, for example, enhancing the flux to and pool of butryl-CoA while minimizing the pool of acetyl-CoA.
In certain embodiments, the present invention provides methods for enhancing butanol production from a bacterial strain. The present invention is not limited to particular methods for enhancing butanol production from a bacterial strain. In some embodiments, the methods involve enhancing butyryl-CoA activity and diminishing acetyl-CoA activity in the bacterial strain for purposes of obtaining increased butanol yield.
The methods are not limited to a particular type of bacterial strain. In some embodiments, the bacterial strain is Clostridium acetobutylicum.
The methods are not limited to a particular manner of enhancing of butyryl-CoA activity. In some embodiments, enhancing of butyryl-CoA activity is accomplished through overexpression of a bifunctional alcohol/aldehyde dehydrogenase gene. The methods are not limited to a particular bifunctional alcohol/aldehyde dehydrogenase gene. Indeed, examples of bifunctional alcohol/aldehyde dehydrogenase genes include the alcohol/aldehyde dehydrogenase (aad) gene.
The methods are not limited to a particular manner of diminishing acetyl-CoA activity. In some embodiments, diminishing acetyl-CoA activity is accomplished through targeting transcripts of enzymes in the acetone formation pathway with antisense RNA. In some embodiments, the antisense RNA is ctfB antisense RNA. In some embodiments, the diminishing of acetyl-CoA activity is accomplished through overexpression of a thiolase gene.
The methods are not limited to a particular manner of regulating overexpession of genes and/or antisense RNA expression. In some embodiments, such regulation is accomplished via a promoter expressed during active cell growth. Examples of promoters expressed during active cell growth include, but are not limited to, a phosphotranbutyrylase (ptb) promoter, a phosphotransacetylase (pta) promoter, and a thiolase (thl) promoter. Any suitable regulatable (e.g., inducible/reproducible) promoter may be used.
In some embodiments, increased butanol and reduced ethanol production in Clostridium acetobutylicum is accomplished through overexpression of the alcohol/aldehyde dehydrogenase gene and the thiolase gene.
In some embodiments, increased butanol and ethanol production in Clostridium acetobutylicum is accomplished through overexpression of the alcohol/aldehyde dehydrogenase gene and through inhibition of acetyl-CoA activity with ctfB antisense RNA.
Embodiments of the present invention provides systems and methods utilizing Clostridium acetobutylicum and ME techniques for alcohol formation and selectivity yields. In some embodiments, the present invention provides, for example, systems and methods to accelerate and enhance alcohol (e.g., butanol) production and selectivity in organisms (e.g., Clostridium acetobutylicum) by, for example, using different combinations of higher aldehyde and alcohol dehydrogenases and/or thiolase expression combined with, for example, CoA transferase downregulation and also by metabolic engineering strategies aimed at, for example, enhancing the flux to and pool of butryl-CoA while minimizing the pool of acetyl-CoA.
Recent advances in molecular biology and metabolic engineering (ME) techniques involving butyric-acid clostridia offer an opportunity to re-establish acetone, butanol and ethanol (ABE) fermentation as an economically viable process. For example, Clostridium acetobutylicum is a model and prototypical organism for the production of such commodity chemicals (e.g., acetone, butanol, ethanol). In particular, Clostridium acetobutylicum is a model and prototypical organism for the production of butanol, which has, for example, emerged as an important new biofuel. The genome of C. acetobutylicum has been sequenced and annotated (Nolling J, et al., 2001, Journal of Bacteriology 183(6):4823-4838; herein incorporated by reference in its entirety), and methods for genetic deletions (Harris L M, et al., 2002, Journal of Bacteriology 184 (13):3586-3597; Heap J T, et al., 2007, J Microbiol Methods 70(3):452-64; Shao L, et al., 2007, Cell Res 170 1):963-5; each herein incorporated by reference in its entireties) and gene overexpression (Mermelstein L D, et al., 1993, Appl Environ Microbiol 59(4): 107710-81; herein incorporated by reference in its entirety) developed. Furthermore, genome-scale microarray-based transcriptional analyses (Alsaker K V, et al., 2005, J Bacteriol 187(20):7103-18; Alsaker K, et al., 2005, Biotechnology and Bioprocess Engineering 10(5):432-443; Alsaker K V, et al., 2004, Journal of Bacteriology. 186(7):1959-1971; Tomas C A, et al., 2003, Journal of Bacteriology 185(15):4539-4547; Tomas C A, et al., 2003, Journal of Bacteriology 186(7):2006-2018; each herein incorporated by reference in its entirety) have illuminated a complex metabolism, thus allowing the development of precise ME strategies (e.g., through genetic modification strategies).
High butanol selectivity and titers in the ABE fermentation are current obstacles for an economical industrial process. Butanol is a valuable product, and thus minimalized production of all other products is desirable. Ethanol is an additional product that may be desirable as a co-product in the context of biofuel production. ABE batch fermentation is characterized by an acidogenic phase and a solventogenic phase. Initially, the cultures produce the organic acids butyrate and acetate, which lower the culture pH. In the solventogenic phase, the culture produces butanol, acetone, and ethanol. Butyrate and acetate are partially re-assimilated to produce solvents, thus raising the pH of the culture. The trigger responsible for the switch from acid to solvent formation (e.g., known as solventogenesis) has been studied, but the exact mechanism for this change remains unknown. The external pH is known to affect solventogenesis and product formation (Husemann M H W, et al., 1988, Biotechnology and Bioengineering 32(7): 843-852; herein incorporated by reference in its entirety). Recent evidence correlates increases of butyryl-phosphate (BuP) concentration with the onset of solvent formation and suggests that BuP performs a role in the regulation of solvent initiation (Zhao Y S, et al., 2005, Appl. Environ Microb. 71(1):530-537; herein incorporated by reference in its entirety).
In wild-type C. acetobutylicum fermentations, final acetone concentrations are typically one-half the final levels of butanol. Initial efforts to increase the selectivity of butanol to acetone used antisense RNA (asRNA) technology targeting the transcripts of enzymes in the acetone formation pathway (see
Experiments conducted during the course of development of embodiments for the present invention demonstrated ME strategies resulting in enhanced butanol formation and selectivity and, significantly, accelerated butanol production. In particular, experiments demonstrated that regulation of fluxes around the two critical nodes of butyryl-CoA and acetyl-CoA (
Accordingly, in some embodiments, the present invention provides methods for enhancing alcohol formation (e.g., ethanol and butanol) from a bacteria strain (e.g., Clostridium acetobutylicum). The present invention is not limited to particular methods for enhancing and acceleration alcohol production from a bacterial strain (e.g., a solventogenic clostridium strain). In some embodiments, the methods comprise enhancing butyryl-CoA activity and diminishing acetyl-CoA activity.
The present invention is not limited to a particular bacteria strain. In some embodiments, the bacteria strain is E. coli. In some embodiments, the bacterial strain is a solventogenic clostridium strain. In some embodiments, the solventogenic clostridium strain is Clostridium acetobutylicum.
The present invention is not limited to a particular method for enhancing butyryl-CoA activity. In some embodiments, enhancement of butyryl-CoA activity is achieved through overexpression of a bifunctional alcohol/aldehyde dehydrogenase gene responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA. The present invention is not limited to a particular bifunctional alcohol/aldehyde dehydrogenase gene responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA. Examples include, but are not limited to, CAP0162 from the C. acetobutylicum genome, CAP0035 C. acetobutylicum genome, CAC3298 C. acetobutylicum genome, CAC3299 C. acetobutylicum genome, CAC3292 C. acetobutylicum genome, and CAP0059 C. acetobutylicum genome. In some embodiments, the gene responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA is alcohol/aldehyde dehydrogenase (aad) gene.
The present invention is not limited to a particular method for diminishing (e.g., inhibiting, reducing) acetyl-CoA activity. In some embodiments, diminishing acetyl-CoA activity is accomplished through targeting the transcripts of enzymes in the acetone formation pathway (see, e.g.,
In some embodiments, overexpression of a bifunctional alcohol/aldehyde dehydrogenase gene responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA (e.g., the aad gene) is regulated via a promoter expressed during active cell growth. In some embodiments, asRNA targeting enzymes in the acetone formation pathway is regulated via a promoter expressed during active cell growth. In some embodiments, a promoter expressed during active cell growth is a phosphotranbutyrylase (ptb) promoter (e.g., of the ptb-buk operon coding two enzymes responsible for butyrate production from butyryl-CoA; see, e.g.,
In some embodiments, the methods for obtaining enhanced alcohol formation is further accomplished through overexpressing one or more genes coding for proteins responsible for butyryl-CoA formation from acetoacetyl-CoA. The methods are not limited to particular genes coding for proteins responsible for butyryl-CoA formation from acetoacetyl-CoA. In some embodiments, genes coding for proteins responsible for butyryl-CoA formation from acetoacetyl-CoA include, but are not limited to, hbd, etfA, etfB, bcd, and cro. In some embodiments, overexpression of one or more genes coding for proteins responsible for butyryl-CoA formation from acetoacetyl-CoA is regulated via one or more promoters expressed during active cell growth (e.g., ptb, pta, thl).
In some embodiments, the methods for obtaining enhanced alcohol production further involve inhibition of ethanol production so as to obtain higher butanol yield. The methods are not limited to a particular manner of inhibiting ethanol production so as to obtain higher butanol yield. In some embodiments, inhibition of ethanol production so as to obtain higher butanol yield is accomplished through downregulation and/or knockout of pyruvate decarboxylase (PDC). In some embodiments, inhibition of ethanol production so as to obtain higher butanol yield is accomplished through overexpression of thiolase (thl) gene. Indeed, in some embodiments, the methods further comprise overexpression of any suitable thiolase gene/protein to enhance the flux from acetyl-CoA to acetoacetyl-CoA and thus minimize the acetylCoA pool. In some embodiments, the methods employ suitable thiolase genes which have been protein engineered by standard methods to generate a thiolase gene with an extremely small Km value for acetyl-CoA in order to drive the acetyl-CoA to acetoacetyl-CoA faster and lower acetyl CoA intracellular pools and thus further minimize the acetyl-CoA pool and thus minimize ethanol production.
The list of bacterial strains and plasmids are in Table I.
C. acetobutylicum
E. coli
aCmr, chloramphenicol resistance gene; ptb, phosphotransbutyrylase gene; aad, alcohol/aldehyde dehydrogenase gene; ctfB, CoA transferase subunit B gene; thl, thiolase gene; adc, acetoacetate decarboxylase gene
bATCC, American Tissue Culture Collection, Rockville, MD
ccontans the following: ampicillin resistance gene; macrolide, lincosimide, and streptogramin B resistance gene: repI, pIM13 Gram-positive origin of replication; ColE1 origin of replication
E. coli strains were grown aerobically at 37° C. and 200 rpm in liquid LB media or solid LB with agar (1.5%) media supplemented with the appropriate antibiotics (ampicillin at 50 μg/mL or chloramphenicol at 35 μg/mL). Frozen stocks were made from 1 mL overnight culture resuspended in LB containing 15% glycerol and stored at −85° C. C. acetobutylicum strains were grown anaerobically at 37° C. in an anaerobic chamber (Thermo Forma, Waltham, Mass.). Cultures were grown in liquid CGM (containing 0.75 g KH2PO4, 0.982 g K2HPO4, 1.0 g NaCl, 0.01 g MnSO4, 0.004 g PABA, 0.348 g MgSO4, 0.01 g FeSO4, 2.0 g asparagine, 5.0 g yeast extract, 2.0 g (NH4)2S04, and 80 g glucose, all per liter) media or solid 2×YTG pH 5.8 (containing 16 g Bacto tryptone, 10 g yeast extract, 4 g NaCl, and 5 g glucose, all per liter) plus agar (1.5%) supplemented with antibiotics as necessary (erythromycin at 100 μg/mL in liquid media and 40 μg/mL in solid media, clarithromycin at 75 μg/mL). Cultures were heat shocked at 70-80° C. for 10 minutes prior to enhance solvent production and prevent strain degeneration (Cornillot E, et al., 1997, J. Bacteriol. 179(17):5442-5447; herein incorporated by reference in its entirety). Frozen stocks were made from 10 mL of A600=1.0 culture resuspended in 1 mL CGM containing 15% glycerol and stored at −85° C.
The aad gene (CAP0162) responsible for butanol formation was PCR amplified from C. acetobutylicum genomic DNA using primers aad_fwd and aad_rev to exclude the natural promoter. All primers used in plasmid construction are listed in Table II. The pSOS94 vector was digested with BamHI and EheI and blunt ended to remove the acetone formation genes while leaving the ptb promoter region and the adc terminator. The aad PCR product and the linearized pSOS94 vector were ligated to create p94AAD3. Both pCTFB1AS, containing the ctfB asRNA, and p94AAD3 were digested with SalI to linearize pCTFB1AS and isolate the aad gene with the ptb promoter and adc terminator from p94AAD3. These fragments were ligated together to generate pCASAAD.
The thiolase (thl) gene including the endogenous promoter and terminator regions was amplified from C. acetobutyiicum genomic DNA using primers thl_fwd and thl_rev. Following purification, the PCR product was digested with SalI and EcoRI as was the shuttle vector pIMP1. The digested PCR product was ligated into the pIMP1 shuttle vector to form the plasmid pTHL. The aad gene cassette from p94AAD3 was isolated using a SalI digestion and purified. Plasmid pTHL was SalI digested and ligated with the purified aad gene cassette to generate plasmid pTHLAAD
A revised ctfB asRNA cassette was generated by first inserting a 100 by oligonucleotide into the pIMP1 shuttle vector following digestion with SalI and EcoRI. This oligonucleotide includes the sequence for the adc promoter element with compatible nucleotide overhangs for ligation. The complimentary oligonucleotides p_adc_top and p_adc_bot were first annealed together before ligating into the pIMP1 vector, creating pPADC, which was then digested with EcoRI and NdeI. A second set of complementary oligonucleotides, ctfBas_top and ctfBas_bot, were annealed and ligated to the digested pPADC to form pCAS. The new ctfB asRNA cassette was PCR amplified from this plasmid using primers cas_fwd and cas_rev and ligated into the pTHLAAD plasmid to generate plasmid pSS2.
All plasmids were transformed into Top 10 chemically competent E. coli (Invitrogen, Carlsbad, Calif.). Plasmids were confirmed using sequencing reactions. The plasmids were methylated using E. Coli ER2275 (pAN 1) cells to avoid the natural restriction system of C. acetobutylicum (Mermelstein L D, et al., 1993, Appl Environ Microbiol 59(4): 107710-81; herein incorporated by reference in its entirety). Once methylated, the plasmids were transformed by electroporating C. acetobutylicum wildtype or mutant M5 strains as described (Mermelstein L D, et al., 1992, Biotechnology (NY) 10(2):190-5; herein incorporated by reference in its entirety).
Fermentations were carried out using a BioFlo 110 or BioFlo II (New Brunswick Scientific Co., Edison, N.J.) bioreactor with 4.0 L working volumes. Fermentations used a 10% v/v inoculum of a pre-culture with A600 equal to 0.2. CGM media were supplemented with 0.10% (v/v) antifoam and 75 μg/mL clarithromycin. Fermentations were maintained at constant pH using 6 M NH4OH. Anaerobic conditions were maintained through nitrogen sparging. Temperature was maintained at 37° C. and agitation was set at 200 rpm. Glucose was restored to the initial concentration (440 mM) in fermentations if glucose levels fell below 200 mM.
Cell density was measured at A600 using a Biomate3 spectrophotometer (Thermo Spectronic, Waltham, Mass.). Samples were diluted as necessary to keep absorbance below 0.40. Supernatant concentrations of glucose, acetone, acetate, acetoin, butyrate, butanol, and ethanol were determined using a high-pressure liquid chromatography system (HPLC) (Waters Corp. Milford, Mass.) (Buday Z, et al., 1990, Enzyme and Microbial Technology 12(1):24-27; herein incorporated by reference in its entirety). Mobile phase of 0.15 mM H2S04 at 0.50 mL/min was used with an Aminex HPLC Organic Acid Analysis Column (Biorad, Hercules, Calif.) The column was cooled to 15° C. and samples were run for 55 minutes.
Cell pellets from 3 to 10 mL of culture were incubated at 37° C. for 4 minutes in 200 μL of SET buffer (25% sucrose, 50 mM EDTA pH 80, 50 mM Tris-HCl pH 8.0) with 20 mg/mL lysozyme. 1 mL trizol was added to each sample and stored at −85° C. until purification. 0.5 mL Trizol and 0.2 mL chloroform was added to 0.5 mL RNA sample and centrifuged at 12,000 rpm for 15 minutes. The aqueous phase was collected and added to an equal volume of isopropanol and RNA was precipitated at 12,000 rpm for 10 minutes. 1 ml, ethanol was added to wash the pellet and centrifuged at 9,500 rpm for 4 minutes. Samples were dried and resuspended in 20-100 μL of RNase free water and stored at −85° C.
Reverse transcription of RNA was carried out using random hexamer primers with 500 μM dNTPs, 2.0 μg RNA, 2 μL RNase inhibitor, 2.5 μL reverse transcriptase, and 2.5 μM random hexamers in a total volume of 100 μL (Applied Biosystems). The reaction was incubated at 25° C. for 10 minutes, 48° C. for 30 minutes, followed by inactivation of the enzymes by a five-minute incubation at 95° C. The SYBR green master mix kit (Applied Biosystems) was used for RT-PCR Each PCR contained 1 μL cDNA and 1 μM gene specific pnmers (Table 2) in a total volume of 25 μL. Samples were performed in tnplicate on a BioRad iCycler with the following parameters: 10 minutes at 95° C., forty cycles of 15 sec at 95° C. and 1 minute at 60° C. All genes were normalized to the pullulanase gene (Tomas C A, et al., 2003, Appl. Environ. Microb. 69(8):4951-4965; herein incorporated by reference in its entirety).
Metabolic Flux analysis calculations were per for med using a program developed by Desai et al (Desai R P, et al., 1999, Journal of Biotechnology 71:191-205; herein incorporated by reference in its entirety). Product concentrations from bioreactor experiments were used to generate metabolic fluxes. Error associated with the calculated fluxes is typically less than 10 percent.
ME strategies to enhance butanol formation and selectivity and accelerate butanol production were explored. The regulation of fluxes around the two critical nodes of butyryl-CoA and acetyl-CoA was explored (
RNA samples were collected during the fermentations and analyzed for the level of aad expression using Q-RT PCR. Comparing the aad expression between the strains, there exists a nearly ten-fold higher expression of aad in 824(pCASAAD) than in 824(pAADB1) during the first four timepoints (
ptb-Promoter-Driven aad Expression Leads to Higher Cell Densities and Increased, Earlier Butanol Formation
Although the growth rate was similar between all strains, 824(pCASAAD) reached higher cell densities (Table III) than either 824(pAADB1) or 824(pSOS95del).
aAll results shown are average mM concentration from duplicate experiments
bResults are from static flask experiments without pH control
These higher cell densities were attributed to, for example, the lower butyrate concentrations observed in the 824(pCASAAD) strain (
Metabolic Flux Analysis of the Three Strains Supports the Limiting Role of Butyryl-CoA and Acetyl-CoA for Butanol Vs. Ethanol Production, Respectively
Using a previously developed model (Desai R P, et al., 1999, Appl Environ Microbiol 65(3):936-45; herein incorporated by reference in its entirety), the fluxes of 824(pAADB1) and 824(pCASAAD) were calculated and normalized both for differences in lag times of growth and cell density. First, the core carbon fluxes GLY 1, GLY 2, thiolase and BYCA, and the H2 formation flux were largely similar among the three strains (except for the first 3-4 normalized hours in strain 824(pCASAAD), which were lower, likely due to the metabolic burden of the early AAD overexpression), and thus unaffected by the genetic modifications, which is theoretically expected and a desirable finding. The butanol and ethanol formation fluxes show significantly higher values early in 824(pCASAAD) than in 824(pAADB1) or the plasmid control. This is consistent with the observation that the FDNH fluxes (NADH2 production from reduced ferredoxin coupled to the GLY 2 flux (
The butyrate formation flux is particularly low in 824(pCASAAD), thus demonstrating, for example, that the strategy for channeling butyryl-CoA from butyrate to butanol formation by the early and strong aad overexpression has worked as anticipated. Due to the low butyrate formation, butyrate uptake is much lower in 824(pCASAAD). Acetate formation is also sustained better and longer in 824(pAADB1) than in 824(pCASAAD) and the plasmid-control strain, and this is consistent with the deduced longer sustained acetyl-CoA pool that sustains much longer a high ethanol flux. Comparing the acid uptake fluxes with the acetone formation flux, it is evident that acetone is produced from the uptake of acetate, as the acetate uptake flux is 10-fold higher than the butyrate uptake flux in both 824(pAADB1) and 824(pCASAAD). Acetone formation is also sustained longer in 824(pAADB1) than in 824(pCASAAD), but both strains show the anticipated lower acetone fluxes compared to the plasmid-control strain as a result of the asRNA downregulation of the acetone-formation enzyme CoAT (
The data discussed above (
To determine if low THL levels were limiting the conversion of acetyl-CoA to butyryl-CoA in the wild-type strain without AAD over expression, the thl gene including its endogenous promoter was amplified from genomic DNA and ligated into the pIMP1 shuttle vector to create plasmid pTHL. Following the transformation of this plasmid into the wild-type strain, pH controlled bioreactors were used to characterize the strain. The metabolism of the 824(pTHL) is characterized by initial levels of high acid production, typical in clostridial fermentations, but there is only very limited acid reassimilation (Table III). Along with the elevated levels of acid production, there is a dramatic decrease in the levels of solvents produced. Additionally, there is a sharp decrease in the cell density of the culture and a plateau of the glucose uptake just a few hours following the peak butyrate production. This indicates, for example, that the cells cannot reassimilate butyrate promptly and the solvent genes cannot be induced to respond to the butyrate production, which leads to growth inhibition. It is hypothesized that, for example, aad overexpression using pptb would promote early butanol production and a means for preventing the accumulation of inhibitory butyrate concentrations.
Overexpression of AAD using pptb was analyzed with (strain 824(pTHLAAD)) and without (strain 824(pPTBAAD)) thl overexpression, and the fermentation data from the two strains are summarized in Table III. As a result of AAD overexpression, ethanol levels in 824(pPTBAAD) increased to 76 mM, more than three times the wild-type production. Additionally butyrate was nearly completely re-assimilated by this strain, while the final butanol titer was 160 mM. Acetate production in 824(pPTBAAD) was also very high reaching final levels of 124 mM. With the addition of THL overexpression, 824(pTHLAAD) shows a significant shift in product formation compared to 824(pPTBAAD). Ethanol production is reduced from 76 mM in 824(pPTBAAD) to 28 mM in 824(pTHLAAD). Acetate formation in 824(pTHLAAD) is also reduced to nearly half the level of 824(pPTBAAD). Butanol is produced at similar levels in both strains while THL overexpression causes a small increase in butyrate formation. Acetone levels were about 40% higher in 824(pTHLAAD) compared to 824(pPTBAAD).
Comparing the profiles of the different fluxes (
thl overexpression achieved the goal of reducing the acetylCoA pool and thus reduce the formation of ethanol and acetate. Indeed, in 824(pPTBAAD) the ratio of the concentrations of the two-carbon products (ethanol and acetate) to the four-carbon products (butanol and butyrate) was 0.81. When THL was overexpressed with AAD in 824(pTHLAAD), this ratio more than doubled to 1.79.
A comparison of the fermentation data (Table III) from strains 824(pPTBAAD) and 824(pCASAAD) illustrates the impact of the asRNA CoAT (
Combined Effect of THL and AAD Overexpression with CoAT Downregulation
Plasmid pSS2 (Table I) was constructed to combine THL, AAD (from the pptb) overexpression, and CoAT downregulation by asRNA, but for the latter using the pptb instead of the pthl used in the pCASAAD and pAADB1 plasmids. pH controlled fermentations of strain 824(pSS2) were once again used to characterize the strain in order to compare to the 824(pCASAAD) and 824(pTHLAAD) strains (
A comparison of strains 824(pSS2) and 824(pTHLAAD) demonstrates the impact of CoAT downregulation in the former is expected in that it reduces acetone formation, but unexpected in that it dramatically enhances ethanol and acetate formation apparently due to an increased acetyl-CoA pool. A similar conclusion is drawn following a comparison between strains 824(pPTBAAD) and 824(pCASAAD) (Table III): CoAT downregulation enhances dramatically ethanol formation but is accompanied by a lower final acetate production. pCASAAD has much higher ethanol and butanol formation fluxes, lower rTHL fluxes, dramatically lower acetate (rACUP) and butyrate (rBYUP) uptake fluxes, altered rFDNH, and altered acetate formation fluxes (higher early, lower later), all of which point to, for example, altered regulation around the acetyl-CoA node.
The pattern of aad expression was altered by replacing the endogenous promoter with that of ptb, which is responsible for butyrate formation. This caused both earlier and higher expression of aad and had marked effects on the fermentation products (
Metabolic flux analysis showed that the earlier expression of aad resulted in earlier formation of both butanol and ethanol. It also appears that butyryl-CoA depletion leads to the high ethanol yields. Ethanol production becomes significant as butanol production decreases due to reduced availability of butyryl-CoA. As the same enzyme (AAD;
The present application claims the benefit of and priority to pending Provisional Patent Application No. 61/082,753, filed Jul. 22, 2008, the entire disclosure of which is hereby incorporated by reference in its entirety.
This invention was made with government support under BES-0418157 (CUFS #0830-350-A320) awarded by the NSF. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61082753 | Jul 2008 | US |
