scholarly journals Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting Saccharomyces cerevisiae Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Bo Li ◽  
Li Wang ◽  
Ya-Jing Wu ◽  
Zi-Yuan Xia ◽  
Bai-Xue Yang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Transcription Factors ◽  
Ethanol Yield ◽  
Regulation Mechanism ◽  
Lignocellulosic Hydrolysate ◽  
Inhibitor Tolerance ◽  
Xylose Consumption ◽  
Content Type ◽  
Beneficial Effects

ABSTRACT Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of Saccharomyces cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) were revealed during mixed glucose and xylose fermentation. Carbohydrate metabolism pathways were significantly enriched in response to Aa, while pathways of xenobiotic biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, other pathways were activated in response to Aa_Fur, i.e., cofactor and vitamin metabolism and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rates by nearly 50%, while the ethanol yield was enhanced by nearly 8% under acetic acid and furfural stress conditions. Co-overexpression of Haa1p and Tye7p resulted in a 59% increase in xylose consumption rate and a 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed acetic acid and furfural. IMPORTANCE Inhibitor tolerance is essential for S. cerevisiae when fermenting lignocellulosic hydrolysate with various inhibitors, including weak acids, furans, and phenols. The details regarding how xylose-fermenting S. cerevisiae strains respond to multiple inhibitors during fermenting mixed glucose and xylose are still unknown. This study revealed the transcriptional regulation mechanism of an industrial xylose-fermenting S. cerevisiae strain in response to acetic acid and furfural. The transcription factor Haa1p was found to be involved in both acetic acid and furfural tolerance. In addition to Haa1p, four other transcription factors, Hap4p, Yox1p, Tye7p, and Mga1p, were identified as able to improve the resistance of yeast to these two inhibitors. This study underscores the feasibility of uncovering effective transcription factors for constructing robust strains for lignocellulosic bioethanol production.

scholarly journals Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

2012 ◽  
Vol 78 (16) ◽  
pp. 5708-5716 ◽  
Author(s):  
Sun-Mi Lee ◽  
Taylor Jellison ◽  
Hal S. Alper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Directed Evolution ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Mutant Enzyme ◽  
Xylose Utilization ◽  
Xylose Consumption ◽  
Content Type ◽  
Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

scholarly journals Deletion ofFPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

2013 ◽  
Vol 79 (10) ◽  
pp. 3193-3201 ◽  
Author(s):  
Na Wei ◽  
Haiqing Xu ◽  
Soo Rin Kim ◽  
Yong-Su Jin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Enzymatic Reactions ◽  
Xylose Metabolism ◽  
Xylose Consumption ◽  
Content Type ◽  
Ethanol Yields

ABSTRACTAccumulation of xylitol in xylose fermentation with engineeredSaccharomyces cerevisiaepresents 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-producingS. cerevisiaestrains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, whenFPS1was 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 thatFPS1deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion ofFPS1decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion ofFPS1resulted 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 inS. cerevisiaeand present a new gene deletion target,FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

scholarly journals Saccharomyces cerevisiae Fermentation of 28 Barley and 12 Oat Cultivars

Fermentation ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 59
Author(s):  
Timothy J. Tse ◽  
Daniel J. Wiens ◽  
Jianheng Shen ◽  
Aaron D. Beattie ◽  
Martin J. T. Reaney
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Lactic Acid ◽  
Succinic Acid ◽  
Alcoholic Fermentation ◽  
Ethanol Yield ◽  
Cereal Crops ◽  
Fermentation Products ◽  
Barley Cultivars ◽  
Oat Cultivars

As barley and oat production have recently increased in Canada, it has become prudent to investigate these cereal crops as potential feedstocks for alcoholic fermentation. Ethanol and other coproduct yields can vary substantially among fermented feedstocks, which currently consist primarily of wheat and corn. In this study, the liquified mash of milled grains from 28 barley (hulled and hull-less) and 12 oat cultivars were fermented with Saccharomyces cerevisiae to determine concentrations of fermentation products (ethanol, isopropanol, acetic acid, lactic acid, succinic acid, α-glycerylphosphorylcholine (α-GPC), and glycerol). On average, the fermentation of barley produced significantly higher amounts of ethanol, isopropanol, acetic acid, succinic acid, α-GPC, and glycerol than that of oats. The best performing barley cultivars were able to produce up to 78.48 g/L (CDC Clear) ethanol and 1.81 g/L α-GPC (CDC Cowboy). Furthermore, the presence of milled hulls did not impact ethanol yield amongst barley cultivars. Due to its superior ethanol yield compared to oats, barley is a suitable feedstock for ethanol production. In addition, the accumulation of α-GPC could add considerable value to the fermentation of these cereal crops.

scholarly journals Cofermentation of Cellobiose and Galactose by an Engineered Saccharomyces cerevisiae Strain

2011 ◽  
Vol 77 (16) ◽  
pp. 5822-5825 ◽  
Author(s):  
Suk-Jin Ha ◽  
Qiaosi Wei ◽  
Soo Rin Kim ◽  
Jonathan M. Galazka ◽  
Jamie Cate ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Neurospora Crassa ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Content Type ◽  
Marine Plant ◽  
Cellodextrin Transporter ◽  
Engineered Saccharomyces Cerevisiae

ABSTRACTWe demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineeredSaccharomyces cerevisiaestrain expressing genes coding for cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) fromNeurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.

scholarly journals Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

2016 ◽  
Vol 82 (7) ◽  
pp. 2156-2166 ◽  
Author(s):  
Yingying Chen ◽  
Lisa Stabryla ◽  
Na Wei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Metabolic Engineering ◽  
Acid Stress ◽  
Acid Resistance ◽  
Biofuel Production ◽  
Gene Target ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

scholarly journals Leveraging Genetic-Background Effects in Saccharomyces cerevisiae To Improve Lignocellulosic Hydrolysate Tolerance

2016 ◽  
Vol 82 (19) ◽  
pp. 5838-5849 ◽  
Author(s):  
Maria Sardi ◽  
Nikolay Rovinskiy ◽  
Yaoping Zhang ◽  
Audrey P. Gasch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Tolerance ◽  
Genetic Background ◽  
Biofuel Production ◽  
Gene Overexpression ◽  
Lignocellulosic Hydrolysate ◽  
Content Type ◽  
Cellular Targets ◽  
Specific Effects ◽  
New Genes

ABSTRACTA major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because the mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variations in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene overexpression in differentSaccharomyces cerevisiae(yeast) strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six differentS. cerevisiaestrains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to identify common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene overexpression in three different strains, we revealed the breadth of the background-specific effects of gene fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms.IMPORTANCERecent studies on natural variation withinSaccharomyces cerevisiaehave uncovered substantial phenotypic diversity. Here, we took advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated the physiological states of cells when exposed to this stress. We also explored the strain-specific effects of gene overexpression to further identify strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

scholarly journals Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

2009 ◽  
Vol 75 (11) ◽  
pp. 3818-3822 ◽  
Author(s):  
Akinori Matsushika ◽  
Hiroyuki Inoue ◽  
Seiya Watanabe ◽  
Tsutomu Kodaki ◽  
Keisuke Makino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Yield ◽  
Xylitol Dehydrogenase ◽  
Wood Chips ◽  
Bioethanol Production ◽  
Xylose Consumption ◽  
Acid Hydrolysate ◽  
A Genome ◽  
Dehydrogenase Gene ◽  
Flocculent Yeast

ABSTRACT The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP+-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

scholarly journals Xylulokinase Overexpression in Two Strains ofSaccharomyces cerevisiae Also Expressing Xylose Reductase and Xylitol Dehydrogenase and Its Effect on Fermentation of Xylose and Lignocellulosic Hydrolysate

2001 ◽  
Vol 67 (9) ◽  
pp. 4249-4255 ◽  
Author(s):  
Björn Johansson ◽  
Camilla Christensson ◽  
Timothy Hobley ◽  
Bärbel Hahn-Hägerdal
Keyword(s):  
Ethanol Yield ◽  
Xylose Reductase ◽  
Xylitol Dehydrogenase ◽  
Host Strain ◽  
Lignocellulosic Hydrolysate ◽  
Xylose Consumption ◽  
Sugar Mixture ◽  
Pentose Sugar ◽  
Ethanol Yields ◽  
Ethanol Producer

ABSTRACT Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) andXYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The geneXKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect ofXKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressingXYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate.XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.

scholarly journals Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

2002 ◽  
Vol 68 (4) ◽  
pp. 1604-1609 ◽  
Author(s):  
Marie Jeppsson ◽  
Björn Johansson ◽  
Bärbel Hahn-Hägerdal ◽  
Marie F. Gorwa-Grauslund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Xylitol Production ◽  
Xylose Consumption ◽  
Xylitol Yield ◽  
Pathway Flux

ABSTRACT In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD+. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g−1) and the lowest xylitol yield (0.05 g g−1) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.

Sign in / Sign up

Export Citation Format

Share Document