Overexpressing CCW12 in Saccharomyces cerevisiae enables highly efficient ethanol production from lignocellulose hydrolysates.

A Saccharomyces cerevisiae strain CCW12OE was constructed by overexpressing CCW12 in a previously reported strain WXY70 harboring six xylose utilization genes. CCW12OE produced an optimal ethanol yield of 98.8% theoretical value within 48 h in a simulated corn stover hydrolysate. CCW12OEwas comprehensively evaluated for ethanol production in Miscanthus, maize and corncob hydrolysates, among which a 96.1% theoretical value was achieved within 12 h in corncob hydrolysates. Under normal growth conditions, CCW12OE did not display altered cell morphology; however, in the presence of acetate, CCW12OE maintained relatively intact cell structure and increased cell wall thickness by nearly 50%, while WXY70 had abnormal cell morphology and reduced cell wall thickness by nearly 50%. Besides, CCW12OE had higher fermentation capacity than that of WXY70 in undetoxified and detoxified hydrolysates with both aerobic and anaerobic conditions, demonstrating that CCW12 overexpression alone exhibits improved stress resistance and better fermentation performance.

Engineered Polyploid Yeast Strains Enable Efficient Xylose Utilization and Ethanol Production in Corn Hydrolysates.

The reported haploid Saccharomyces cerevisiae strain F106 can utilize xylose for ethanol production. After a series of XR and/or XDH mutations were introduced into F106, the XR-K270R mutant was found to outperform others. The corresponding haploid, diploid, and triploid strains were then constructed and their fermentation performance was compared. Strains F106-KR and the diploid produced an ethanol yield of 0.45 and 0.48 g/g total sugars, respectively, in simulated corn hydrolysates within 36 h. Using non-detoxicated corncob hydrolysate as the substrate, the ethanol yield with the triploid was approximately sevenfold than that of the diploid at 40°C. After a comprehensive evaluation of growth on corn stover hydrolysates pretreated with diluted acid or alkali and different substrate concentrations, ethanol yields of the triploid strain were consistently higher than those of the diploid using acid-pretreatment. These results demonstrate that the yeast chromosomal copy number is positively correlated with increased ethanol production under our experimental conditions.

Metabolic and Evolutionary Engineering of Diploid Yeast for the Production of First- and Second-Generation Ethanol.

Despite a growing preference for second-generation (2G) ethanol in industries, its application is severely restricted owing to a major obstacle of developing a suitable yeast strain for fermentation using feedstock biomasses. In this study, a yeast strain, Saccharomyces cerevisiae A31Z, for 2G bioethanol production was developed from an industrial strain, Angel, using metabolic engineering by the incorporation of gene clusters involved in the xylose metabolism combined with adaptive evolution for evolving its anti-inhibitory properties. This strain outcompeted its ancestors in xylose utilization and subsequent ethanol production and manifested higher tolerance against common inhibitors from lignocellulosic hydrolysates, and also it lowered the production of glycerol by-product. Furthermore, A31Z outperformed in ethanol production using industrial hydrolysate from dried distillers grains with solubles and whole corn. Overall, this study provided a promising path for improving 2G bioethanol production in industries using S. cerevisiae.

Overexpression of SFA1 in engineered Saccharomyces cerevisiae to increase xylose utilization and ethanol production from different lignocellulose hydrolysates.

Here, an engineered Saccharomyces cerevisiae strain SFA1OE was constructed by overexpressing SFA1 in a reported WXY70 with effective six-gene clusters. Under simulated maize hydrolysate, SFA1OE produced an ethanol yield of 0.492 g/g totalsugars within 48 h. The productivity of SFA1OE was comprehensively evaluated in typical hydrolysates from stalks of maize, sweet sorghum, wheat and Miscanthus. Within 48 h, SFA1OE achieved an ethanol yield of 0.489 g/g totalsugars in the optimized hydrolysate of alkaline-distilled sweet sorghum bagasse derived from Advanced Solid-State Fermentation process. By crossing SFA1OE with a DQ1-derived haploid strain, we obtained an evolved diploid strain SQ-2, exhibiting improved ethanol production and thermotolerance. This study demonstrates that overexpressing SFA1 enables efficient fermentation performance in different lignocellulosic hydrolysates, especially in the hydrolysate of alkaline-distilled sweet sorghum bagasse. The increased cellulosic bioethanol production of SFA1OE provides a promising platform for efficient biorefineries.