Increase In Ethanol Yield Research Articles

Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum.

Recombinant Corynebacterium glutamicum harboring genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) can produce ethanol under oxygen deprivation. We investigated the effects of elevating the expression levels of glycolytic genes, as well as pdc and adhB, on ethanol production. Overexpression of four glycolytic genes (pgi, pfkA, gapA, and pyk) in C. glutamicum significantly increased the rate of ethanol production. Overexpression of tpi, encoding triosephosphate isomerase, further enhanced productivity. Elevated expression of pdc and adhB increased ethanol yield, but not the rate of production. Fed-batch fermentation using an optimized strain resulted in ethanol production of 119 g/L from 245 g/L glucose with a yield of 95% of the theoretical maximum. Further metabolic engineering, including integration of the genes for xylose and arabinose metabolism, enabled consumption of glucose, xylose, and arabinose, and ethanol production (83 g/L) at a yield of 90 %. This study demonstrated that C. glutamicum has significant potential for the production of cellulosic ethanol.

Synthetic biology applications in industrial microbiology

Synthetic biology applications in industrial microbiology

Improvements of tolerance to stress conditions by genetic engineering in Saccharomyces cerevisiae during ethanol production.

Saccharomyces cerevisiae, industrial yeast isolate, has been of great interest in recent years for fuel ethanol production. The ethanol yield and productivity depend on many inhibitory factors during the fermentation process such as temperature, ethanol, compounds released as the result of pretreatment procedures, and osmotic stress. An ideal strain should be able to grow under different stress conditions occurred at different fermentation steps. Development of tolerant yeast strains can be achieved by reprogramming pathways supporting the ethanol metabolism by regulating the energy balance and detoxicification processes. Complex gene interactions should be solved for an in-depth comprehension of the yeast stress tolerance mechanism. Genetic engineering as a powerful biotechnological tool is required to design new strategies for increasing the ethanol fermentation performance. Upregulation of stress tolerance genes by recombinant DNA technology can be a useful approach to overcome inhibitory situations. This review presents the application of several genetic engineering strategies to increase ethanol yield under different stress conditions including inhibitor tolerance, ethanol tolerance, thermotolerance, and osmotolerance.

Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase.

BackgroundThe production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity.ResultsIntact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production.ConclusionOverexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.

Enhanced fermentability of poplar by combination of alkaline peroxide pretreatment and semi-simultaneous saccharification and fermentation.

To improve ethanol productivity with few inhibitors generated, a novel process of combined alkaline peroxide (AP) pretreatment and semi-simultaneous saccharification and fermentation (SSSF) was developed in this work. Pretreatment with 10% (g H2O2/g wood) H2O2 at 160°C for 2h followed by SSSF was found to be the optimal combination with remarkably increased ethanol yield. The proposed process resulted in 63.1% of ethanol yield, which was about five times more than that of the untreated sample that was processed using conventional simultaneous saccharification and fermentation (SSF). The efficient conversion was ascribed to the high delignification efficiency (64.9%) of AP pretreatment, which led to incompact structure and generation of fewer inhibitors during SSSF (c. 6g/L of lactic acid) than SSF (c. 10 g/L of lactic acid). This combined approach was proved to be an effective method for the promotion of the bioconversion of lignocellulosic materials.

Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum

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) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.

Helically agitated mixing in dry dilute acid pretreatment enhances the bioconversion of corn stover into ethanol.

BackgroundDry dilute acid pretreatment at extremely high solids loading of lignocellulose materials demonstrated promising advantages of no waste water generation, less sugar loss, and low steam consumption while maintaining high hydrolysis yield. However, the routine pretreatment reactor without mixing apparatus was found not suitable for dry pretreatment operation because of poor mixing and mass transfer. In this study, helically agitated mixing was introduced into the dry dilute acid pretreatment of corn stover and its effect on pretreatment efficiency, inhibitor generation, sugar production, and bioconversion efficiency through simultaneous saccharification and ethanol fermentation (SSF) were evaluated.ResultsThe overall cellulose conversion taking account of cellulose loss in pretreatment was used to evaluate the efficiency of pretreatment. The two-phase computational fluid dynamics (CFD) model on dry pretreatment was established and applied to analyze the mixing mechanism. The results showed that the pretreatment efficiency was significantly improved and the inhibitor generation was reduced by the helically agitated mixing, compared to the dry pretreatment without mixing: the ethanol titer and yield from cellulose in the SSF reached 56.20 g/L and 69.43% at the 30% solids loading and 15 FPU/DM cellulase dosage, respectively, corresponding to a 26.5% increase in ethanol titer and 17.2% increase in ethanol yield at the same fermentation conditions.ConclusionsThe advantage of helically agitated mixing may provide a prototype of dry dilute acid pretreatment processing for future commercial-scale production of cellulosic ethanol.

Overexpression of NADH-dependent fumarate reductase improves d-xylose fermentation in recombinant Saccharomyces cerevisiae

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD+POS5Δ17 resulted in 60 and 23% increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production

Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD+-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-013-1311-5) contains supplementary material, which is available to authorized users.

Prediction of Weak Acid Toxicity inSaccharomyces cerevisiaeUsing Genome-Scale Metabolic Models

The use of lignocellulosic biomass is critical for the economic production of transportation fuels and chemicals in renewable bioprocesses. While biomass is an abundant resource, necessary pretreatment to yield fermentable monosaccharides produces toxic compounds that dramatically affect fermentation performance. Weak acids such as acetic acid play an important role in the toxicity of lignocellulosic hydrolysate to Saccharomyces cerevisiae, a commonly used industrial organism. In order to explore the ramifications of weak acid inhibition on cellular metabolism, we adapted a genome-scale metabolic model of S. cerevisiae to describe toxicity of acetic acid by a decoupling mechanism. We evaluated the performance of the model in predicting growth rates and ethanol production characteristics under aerobic and anaerobic cultivations. We found that the model was able to capture the decreased growth during aerobic cultivations in the presence of acetic acid, but was unable to capture the increase in ethanol yield observed. The model was able to predict anaerobic growth rates and ethanol yields; however, at conditions of higher toxicity levels, discrepancies arose. We expect that a model such as this may find application in the optimization of lignocellulose-based bioprocesses in which there exists a critical economic trade-off between neutralization costs and product yields.

3′ Truncation of the GPD1 Promoter in Saccharomyces cerevisiae for Improved Ethanol Yield and Productivity

Glycerol is a major by-product in bioethanol fermentation by the yeast Saccharomyces cerevisiae, and decreasing glycerol formation for increased ethanol yield has been a major research effort in the bioethanol field. A new strategy has been used in the present study for reduced glycerol formation and improved ethanol fermentation performance by finely modulating the expression of GPD1 in the KAM15 strain (fps1Δ pPGK1-GLT1 gpd2Δ). The GPD1 promoter was serially truncated from the 3' end by 20 bp to result in a different expression strength of GPD1. The two engineered promoters carrying 60- and 80-bp truncations exhibited reduced promoter strength but unaffected osmostress response. These two promoters were integrated into the KAM15 strain, generating strains LE34U and LE35U, respectively. The transcription levels of LE34U and LE35U were 37.77 to 45.12% and 21.34 to 24.15% of that of KAM15U, respectively, depending on osmotic stress imposed by various glucose concentrations. In very high gravity (VHG) fermentation, the levels of glycerol for LE34U and LE35U were reduced by 15.81% and 30.66%, respectively, compared to KAM15U. The yield and final concentration of ethanol for LE35U were 3.46% and 0.33% higher, respectively, than those of KAM15U. However, fermentation rate and ethanol productivity for LE35U were reduced. On the other hand, the ethanol yield and final concentration for LE34U were enhanced by 2.28% and 2.32%, respectively, compared to those of KAM15U. In addition, a 2.31% increase in ethanol productivity was observed for LE34U compared to KAM15U. These results verified the feasibility of our strategy for yeast strain development.

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD(+)/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

Rational and Evolutionary Engineering Approaches Uncover a Small Set of Genetic Changes Efficient for Rapid Xylose Fermentation in Saccharomyces cerevisiae

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

Modeling and dynamic optimization of fuel-grade ethanol fermentation using fed-batch process

This paper investigates optimization of operational strategies of an industrial ethanol fermentation process. One of the challenges associated with this type or process is that most of the measurements are only taken sporadically, thereby complicating process monitoring and optimization. The one exception to this rule involves temperature measurements, which are readily available. However, an existing model of the plant investigated in this paper does not include an energy balance and, accordingly, the temperature measurements cannot be used to estimate model parameters. This paper addresses these deficiencies and proposes modifications to an existing ethanol fermentation model. The proposed changes include the derivation of an energy balance, modification of the reaction kinetics to include additional inhibition terms, and also estimation of model parameters from industrial data. The new model is validated against plant data and then used for optimization of the process operations. It is shown that modifications of the input profiles for the cooling rate and the glucoamylase addition can lead to an approximately 10% increase in ethanol yield. These are promising results, even though these findings will ultimately need to be validated during real plant operations.

Comparative study on the mutated xylose reductase to increase ethanol production in xylose-utilizing Saccharomyces cerevisiae strains

We constructed one wild-type xylose reductase (XR) and eight XR variants in a recombinant industrial Saccharomyces cerevisiae strain along with xylitol dehydrogenase and xylulokinase overexpression. In the current study, we found that K270R XR (hereafter referred to as “XR-1”) appeared to utilize more NADH than NADPH when xylose was the carbon source. After 72h of fermentation, XR-1 produced an ethanol yield of 0.34g/g and a xylitol yield of 0.08g/g from 55g/L xylose, while it gave an ethanol yield of 0.44g/g and a xylitol yield of 0.04g/g under a mixture of 40g/L xylose and 40g/L glucose. Notably, the findings presented here suggest that the XR-1 variant produced approximately 76% more ethanol from xylose than the wild-type XR strain. Comparing with other widely divergent XR mutations, the XR-1(K270R) exhibited improved xylose consumption rate, balanced redox system and increased ethanol yield and production rate when engineered into S. cerevisiae, suggesting the significance of mutations to the XR gene in xylose utilization.

Utilization of concentrate after membrane filtration of sugar beet thin juice for ethanol production

The subject of this study was to investigate the feasibility of the concentrate obtained after membrane ultrafiltration of sugar beet thin juice for ethanol production and selection of fermentation conditions (yeast strain and media supplementation). Resulting concentrate was subjected to batch ethanol fermentation using two strains of Saccharomyces cerevisiae (Ethanol Red and Safdistill C-70). The effect of different forms of media supplementation (mineral salts: (NH4)2SO4, K2HPO4, MgCl2; urea+Mg3(PO4)2 and yeast extract) on the fermentation course was also studied. It was stated that sugar beet juice concentrate is suitable for ethanol production yielding, depending on the yeast strain, ca. 85–87gL−1 ethanol with ca. 82% practical yield and more than 95% of sugars consumption after 72h of fermentation. Nutrients enrichment further increased ethanol yield. The best results were obtained for media supplemented with urea+Mg3(PO4)2 yielding 91.16–92.06gL−1 ethanol with practical yield ranging 84.78–85.62% and full sugars consumption.

Influence of fiber degradation and concentration of fermentable sugars on simultaneous saccharification and fermentation of high-solids spruce slurry to ethanol

BackgroundSaccharification and fermentation of pretreated lignocellulosic materials, such as spruce, should be performed at high solids contents in order to reduce the cost of the produced bioethanol. However, this has been shown to result in reduced ethanol yields or a complete lack of ethanol production. Previous studies have shown inconsistent results when prehydrolysis is performed at a higher temperature prior to the simultaneous saccharification and fermentation (SSF) of steam-pretreated lignocellulosic materials. In some cases, a significant increase in overall ethanol yield was reported, while in others, a slight decrease in ethanol yield was observed. In order to investigate the influence of prehydrolysis on high-solids SSF of steam-pretreated spruce slurry, in the present study, the presence of fibers and inhibitors, degree of fiber degradation and initial fermentable sugar concentration has been studied.ResultsSSF of whole steam-pretreated spruce slurry at a solids content of 13.7% water-insoluble solids (WIS) resulted in a very low overall ethanol yield, mostly due to poor fermentation. The yeast was, however, able to ferment the washed slurry and the liquid fraction of the pretreated slurry. Performing prehydrolysis at 48°C for 22 hours prior to SSF of the whole pretreated slurry increased the overall ethanol yield from 3.9 to 62.1%. The initial concentration of fermentable sugars in SSF could not explain the increase in ethanol yield in SSF with prehydrolysis. Although the viscosity of the material did not appear to decrease significantly during prehydrolysis, the degradation of the fibers prior to the addition of the yeast had a positive effect on ethanol yield when using whole steam-pretreated spruce slurry.ConclusionsThe results of the present study suggest that the increase in ethanol yield from SSF when performing prehydrolysis is a result of fiber degradation rather than a decrease in viscosity. The increased concentration of fermentable sugars at the beginning of the fermentation phase in SSF following prehydrolysis did not affect the overall ethanol yield in the present study.

Trends in global warming and human health impacts related to Brazilian sugarcane ethanol production considering black carbon emissions

Sugarcane produced in Brazil has several environmental advantages. However, burning residues, which leads to GHG and black carbon (BC) emissions, has been used to facilitate manual harvest. BC emissions have a net warming effect and cause health problems. Mechanized harvest without burning is gradually replacing manually harvested burned sugarcane. Global warming potential (GWP) and human health indicators of sugarcane ethanol production in Brazil, in the pre-mechanization (100% burned), current (∼50% burned) and future (100% without burning) scenarios, were calculated. In the past, the GWP of ethanol production was 1.1kgCO2eqL−1 and BC emissions were 32.6kgCO2eqL−1. The human health impact in disability adjusted life years (DALY) was 3.16E−05DALYL−1 ethanol. The current ethanol production process has a GWP 46% smaller, while BC emissions are seven times smaller than before mechanization started. The human health impact is currently 7.72E−06DALYL−1. In the future, with complete mechanization and the integration of first and second generation ethanol, the expected GWP emissions will be 70% smaller, and BC emissions will be 216 times smaller than when all sugarcane was harvested with burning. These results show that ethanol production in Brazil is improving in terms of global warming and human health aspects. Other upstream aspects of ethanol production such as direct and indirect land use change, and downstream impacts such as the emissions of acetaldehydes were not considered in this study, which focused on a major technological shift in residue management in the agricultural phase of sugarcane ethanol production. A broader assessment of the sustainability of ethanol must account for those issues, as well as economic and social aspects. Sugarcane-derived ethanol produced in Brazil has been considered one of the most sustainable biofuels options, but it is essential to identify and promote practices and policies that further improve its production, such as the phase out of pre-harvest sugarcane burning and the increase in ethanol yield per unit of area.

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR–XDH pathway

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD(+) to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50g/L xylose in the presence of 40mM furfural and most efficiently produced ethanol (15.70g/L and 15.24g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.

Cre-lox66/lox71-Based Elimination of Phosphotransacetylase or Acetaldehyde Dehydrogenase Shifted Carbon Flux in Acetogen Rendering Selective Overproduction of Ethanol or Acetate

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H(2). Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p<0.005). Inactivation of pta rendered only 502 mM of ethanol (p<0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p<0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.