Microaerobic conversion of glycerol to ethanol in Escherichia coli.
ABSTRACT: Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.
Project description:Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD<sup>+</sup> during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (<i>adhE</i>) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1?MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in <i>Clostridium ljungdahlii</i> A metabolic experiment using <sup>13</sup>C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases.<b>IMPORTANCE</b> Ethanol production from carbon monoxide (CO) as a carbon and energy source by <i>Clostridium ljungdahlii</i> and "<i>Clostridium autoethanogenum</i>" is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of <i>C. ljungdahlii</i> in the future.
Project description:Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols.
Project description:Acetaldehyde-alcohol dehydrogenase (AdhE) enzymes are a key metabolic enzyme in bacterial physiology and pathogenicity. They convert acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in an anaerobic environment. This two-step reaction is associated to NAD+ regeneration, essential for glycolysis. The bifunctional AdhE enzyme is conserved in all bacterial kingdoms but also in more phylogenetically distant microorganisms such as green microalgae. It is found as an oligomeric form called spirosomes, for which the function remains elusive. Here, we use cryo-electron microscopy to obtain structures of Escherichia coli spirosomes in different conformational states. We show that spirosomes contain active AdhE monomers, and that AdhE filamentation is essential for its activity in vitro and function in vivo. The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling within the filament and for the regulation of enzyme activity.
Project description:Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ?adhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ?adhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.
Project description:Background:Due to its inevitable formation during biodiesel production and its relatively high degree of reduction, glycerol is an attractive carbon source for microbial fermentation processes. However, glycerol is catabolized in a fully respiratory manner by the eukaryotic platform organism Saccharomyces cerevisiae. We previously engineered S. cerevisiae strains to favor fermentative metabolism of glycerol by replacing the native FAD-dependent glycerol catabolic pathway with the NAD-dependent 'DHA pathway'. In addition, a heterologous aquaglyceroporin (Fps1 homolog) was expressed to facilitate glycerol uptake. The current study was launched to scrutinize the formation of S. cerevisiae's natural fermentation product ethanol from glycerol caused by the conducted genetic modifications. This understanding is supposed to facilitate future engineering of this yeast for fermenting glycerol into valuable products more reduced than ethanol. Results:A strain solely exhibiting the glycerol catabolic pathway replacement produced ethanol at concentrations close to the detection limit. The expression of the heterologous aquaglyceroporin caused significant ethanol production (8.5 g L-1 from 51.5 g L-1 glycerol consumed) in a strain catabolizing glycerol via the DHA pathway but not in the wild-type background. A reduction of oxygen availability in the shake flask cultures further increased the ethanol titer up to 15.7 g L-1 (from 45 g L-1 glycerol consumed). Conclusion:The increased yield of cytosolic NADH caused by the glycerol catabolic pathway replacement seems to be a minimal requirement for the occurrence of alcoholic fermentation in S. cerevisiae growing in synthetic glycerol medium. The remarkable metabolic switch to ethanol formation in the DHA pathway strain with the heterologous aquaglyceroporin supports the assumption of a much stronger influx of glycerol accompanied by an increased rate of cytosolic NADH production via the DHA pathway. The fact that a reduction of oxygen supply increases ethanol production in DHA pathway strains is in line with the hypothesis that a major part of glycerol in normal shake flask cultures still enters the catabolism in a respiratory manner.
Project description:Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.
Project description:Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) ?ldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) ?ldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.
Project description:Background:Fed-batch fermentation has been conventionally implemented for the production of lactic acid with a high titer and high productivity. However, its operation needs a complicated control which increases the production cost. Results:This issue was addressed by simplifying the production scheme. Escherichia coli was manipulated for its glycerol dissimilation and d-lactate synthesis pathways and then subjected to adaptive evolution under high crude glycerol. Batch fermentation in the two-stage mode was performed by controlling the dissolved oxygen (DO), and the evolved strain deprived of poxB enabled production of 100 g/L d-lactate with productivity of 1.85 g/L/h. To increase productivity, the producer strain was further evolved to improve its growth rate on crude glycerol. The fermentation was performed to undergo the aerobic growth with low substrate, followed by the anaerobic production with high substrate. Moreover, the intracellular redox of the strain was balanced by fulfillment of the anaerobic respiratory chain with nitrate reduction. Without controlling the DO, the microbial fermentation resulted in the homofermentative production of d-lactate (ca. 0.97 g/g) with a titer of 115 g/L and productivity of 3.29 g/L/h. Conclusions:The proposed fermentation strategy achieves the highest yield based on crude glycerol and a comparable titer and productivity as compared to the approach by fed-batch fermentation. It holds a promise to sustain the continued development of the crude glycerol-based biorefinery.
Project description:UNLABELLED:Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCE:Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth.
Project description:Aldehyde/alcohol dehydrogenases (ADHEs) are bifunctional enzymes that commonly produce ethanol from acetyl-CoA with acetaldehyde as intermediate and play a key role in anaerobic redox balance in many fermenting bacteria. ADHEs are also present in photosynthetic unicellular eukaryotes, where their physiological role and regulation are, however, largely unknown. Herein we provide the first molecular and enzymatic characterization of the ADHE from the photosynthetic microalga Chlamydomonas reinhardtii Purified recombinant ADHE catalyzed the reversible NADH-mediated interconversions of acetyl-CoA, acetaldehyde, and ethanol but seemed to be poised toward the production of ethanol from acetaldehyde. Phylogenetic analysis of the algal fermentative enzyme supports a vertical inheritance from a cyanobacterial-related ancestor. ADHE was located in the chloroplast, where it associated in dimers and higher order oligomers. Electron microscopy analysis of ADHE-enriched stromal fractions revealed fine spiral structures, similar to bacterial ADHE spirosomes. Protein blots showed that ADHE is regulated under oxic conditions. Up-regulation is observed in cells exposed to diverse physiological stresses, including zinc deficiency, nitrogen starvation, and inhibition of carbon concentration/fixation capacity. Analyses of the overall proteome and fermentation profiles revealed that cells with increased ADHE abundance exhibit better survival under dark anoxia. This likely relates to the fact that greater ADHE abundance appeared to coincide with enhanced starch accumulation, which might reflect ADHE-mediated anticipation of anaerobic survival.