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 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 Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

2012 ◽  
Vol 79 (5) ◽  
pp. 1500-1507 ◽  
Author(s):  
Suk-Jin Ha ◽  
Heejin Kim ◽  
Yuping Lin ◽  
Myoung-Uoon Jang ◽  
Jonathan M. Galazka ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Wild Type ◽  
Scheffersomyces Stipitis ◽  
Content Type ◽  
Charged Amino Acids ◽  
Cellodextrin Transporter ◽  
Engineered Strain

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.

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 Efficient Alcoholic Conversion of Glycerol by Engineered Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Takashi Watanabe ◽  
Sadat M. R. Khattab
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotinamide Adenine Dinucleotide ◽  
Batch Fermentation ◽  
Plant Biomass ◽  
Glycerol Oxidation ◽  
Fed Batch ◽  
Fed Batch Fermentation ◽  
Engineered Saccharomyces Cerevisiae ◽  
Biomass Decomposition ◽  
Engineered Strain

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae hinders its use in these methods. Here, we have aimed to develop a complete strategy for the generation of efficient glycerol-converting yeast by modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O2-dependent dynamic shuttle, while abolishing both glycerol phosphorylation and biosynthesis. By following a vigorous glycerol oxidation pathway, the engineered strain increased the conversion efficiency (CE) to up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate > 1 g×L×h, when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain fermented a mixture of glycerol and glucose, producing > 86 g/L bioethanol with 92.8% CE. To our knowledge, this is the highest ever reported titer in this field. Notably, this strategy changed conventional yeast from a non-grower on minimal medium containing glycerol to a fermenting strain with productivity of 0.25-0.5 g×L×h and 84-78% CE, which converted 90% of the substrate to products. Our findings may improve the utilization of glycerol in several eco-friendly biorefinery approaches.

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 Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

2013 ◽  
Vol 80 (3) ◽  
pp. 1002-1012 ◽  
Author(s):  
Shodai Shiroma ◽  
Lahiru Niroshan Jayakody ◽  
Kenta Horie ◽  
Koji Okamoto ◽  
Hiroshi Kitagaki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Strain ◽  
Parent Strain ◽  
Ethanol Yield ◽  
Synthetic Medium ◽  
Nutrient Concentrations ◽  
Content Type ◽  
Fermentative Capacity ◽  
Limiting Nutrient ◽  
Biomass Resources

ABSTRACTSaccharomyces cerevisiaesake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO2production and final ethanol concentration generated by theatg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of theatg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that theatg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.

Improvement of Ethanol Yield from Glycerol via Conversion of Pyruvate to Ethanol in Metabolically Engineered Saccharomyces cerevisiae

2011 ◽  
Vol 166 (4) ◽  
pp. 856-865 ◽  
Author(s):  
Kyung Ok Yu ◽  
Ju Jung ◽  
Ahmad Bazli Ramzi ◽  
Seung Wook Kim ◽  
Chulhwan Park ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Yield ◽  
Engineered Saccharomyces Cerevisiae

scholarly journals Enhanced ethanol production from sugarcane molasses by industrially engineered Saccharomyces cerevisiae via replacement of the PHO4 gene

RSC Advances ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 2267-2276 ◽  
Author(s):  
Renzhi Wu ◽  
Dong Chen ◽  
Shuwei Cao ◽  
Zhilong Lu ◽  
Jun Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Ethanol Yield ◽  
Regulatory Gene ◽  
Sugarcane Molasses ◽  
Fermentation Time ◽  
Fast Growing ◽  
Novel Strategy ◽  
Engineered Saccharomyces Cerevisiae

Replacement of a novel candidate ethanol fermentation-associated regulatory gene, PHO4, from a fast-growing strain through a novel strategy (SHPERM-bCGHR), is hypothesised to shorten fermentation time and enhance ethanol yield from sugarcane molasses.

scholarly journals Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

2015 ◽  
Vol 81 (23) ◽  
pp. 8108-8117 ◽  
Author(s):  
Brooks M. Henningsen ◽  
Shuen Hon ◽  
Sean F. Covalla ◽  
Carolina Sonu ◽  
D. Aaron Argyros ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Ethanol Yield ◽  
Pentose Phosphate ◽  
Wild Type ◽  
Bifidobacterium Adolescentis ◽  
Acetaldehyde Dehydrogenase ◽  
Content Type ◽  
Sugar Fermentation ◽  
Ethanol Yields

ABSTRACTSaccharomyces cerevisiaehas recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering ofS. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase fromBifidobacterium adolescentisand with deletions of glycerol-3-phosphate dehydrogenase genesGPD1andGPD2) consumed 1.9 g liter−1acetate during fermentation of 114 g liter−1glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH fromEntamoeba histolyticaand with overexpression ofACS2andZWF1, we increased acetate consumption to 5.3 g liter−1and raised the ethanol yield to 7% above the wild-type level.

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