关键词: Saccharomyces cerevisiae, Ethanol, Genetic engineering, Adaptive evolution, Xylose uptake, Lignocellulosic hydrolysate

Abstract: Saccharomyces cerevisiae is not naturally capable of efficiently utilizing xylose as a carbon source. When cultured with lignocellulosic hydrolysates containing pretreatment-derived inhibitors, S. cerevisiae suffers from much lower sugar uptake, ethanol yield and fermentation efficiency. Thus, considering efficient xylose conversion into ethanol during non-detoxified hydrolysate culture, genetic engineering and adaptive evolution of S. cerevisiae might be a promising joint strategy for improving xylose uptake and ethanol production. In this study, an inhibitor-tolerant strain S. cerevisiae SPSC01-TAF94 was genetically engineered by overexpressing both xylose transport- and metabolism-related genes (N360F, Ru-xylA, TAL1, TKL1, RKI1 and RPE1), yielding the xylose-utilizing strain TAF94-X, followed by three-stage adaptation in non-detoxified corn stover hydrolysate containing 5 g·L^-1 acetic acid, 0.32 g·L^-1 furfural, 0.17 g·L^-1 HMF and 0.19 g·L^-1 vanillin as the major inhibitors as well as 20, 40 and 60 g·L^-1 xylose adjusted as the major carbon source, respectively. Finally, an active xylose-utilizing and ethanol-producing strain TAF94-X60 was obtained, which achieved 44.9 g·L^-1 ethanol with yield of 0.41 g·g^-1, productivity of 0.62 g·L^-1·h^-1 and xylose consumption rate of 0.42 g·L^-1·h^-1 during hydrolysate culture, compared to those of 36.5 g·L^-1, 0.38 g·g^-1, 0.50 g·L^-1·h^-1 and 0.20 g·L^-1·h^-1 obtained with the control strain TAF94-X. The proposed joint strategy effectively utilizes hydrolyzed sugars while eliminating the need for conventional detoxification or water washing processes, thus enhancing the economic feasibility of large-scale lignocellulosic ethanol production.

Key words: Saccharomyces cerevisiae, Ethanol, Genetic engineering, Adaptive evolution, Xylose uptake, Lignocellulosic hydrolysate