Abstract Saccharomyces cerevisiae strain with excellent xylose-fermenting capacity and inhibitor tolerance is crucial for lignocellulosic ethanol production. In this study, a combined strategy including site-directed mutagenesis, mating, evolutionary engineering, and haploidization was applied to obtain strains with ideal xylose fermentabilities. Haploid industrial strain KFG4-6B was engineered to overexpress endogenous xylulokinase (XK) and heterologous native or mutated xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis. The XR-mutated strain HX57D showed over 12% increase in both xylose consumption rate and ethanol yield compared with the XR-native strain. To improve the xylose uptake, the HX57D-derived diploids were subjected to evolutionary engineering. In comparison with HX57D, evolved diploid Z4X-21-18 achieved 4.5-fold increases in rates of xylose consumption and ethanol production when fermenting xylose. When fermenting mixed sugars, the glucose and xylose uptake rates were 1.4-fold and 8.3-fold, respectively, higher. H18s28, a haploid of Z4X-21-18, enabled a further 10% increase in xylose consumption rate when fermenting xylose only. However, it was inferior to its diploid parent when fermenting mixed sugars. In the presaccharification-simultaneous saccharification and fermentation (P-SSF) of the whole pretreated wheat straw slurry with high contents of multiple inhibitors, Z4X-21-18 produced approximately 42 g/L ethanol with a yield of 0.38 g/g total sugars.

中文翻译：

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通过组合方法构建工业木糖发酵酿酒酵母菌株

摘要 具有优异的木糖发酵能力和抑制剂耐受性的酿酒酵母菌株对木质纤维素乙醇的生产至关重要。在这项研究中，应用包括定点诱变、交配、进化工程和单倍体化在内的组合策略来获得具有理想木糖发酵能力的菌株。单倍体工业菌株 KFG4-6B 被设计为过表达来自树干裂酵母的内源性木酮糖激酶 (XK) 和异源天然或突变木糖还原酶 (XR) 和木糖醇脱氢酶 (XDH)。与 XR 天然菌株相比，XR 突变菌株 HX57D 的木糖消耗率和乙醇产量均增加了 12% 以上。为了提高木糖摄取，HX57D 衍生的二倍体进行了进化工程。与HX57D相比，在发酵木糖时，进化出的二倍体 Z4X-21-18 的木糖消耗率和乙醇产量提高了 4.5 倍。发酵混合糖时，葡萄糖和木糖的吸收率分别高出 1.4 倍和 8.3 倍。H18s28 是 Z4X-21-18 的单倍体，仅发酵木糖时，木糖消耗率进一步提高了 10%。然而，它在发酵混合糖时不如其二倍体亲本。Z4X-21-18在对多重抑制剂含量高的全预处理麦秆浆进行预糖化-同步糖化发酵（P-SSF）时，乙醇产量约为42 g/L，总糖产量为0.38 g/g。葡萄糖和木糖的摄取率分别高出 1.4 倍和 8.3 倍。H18s28 是 Z4X-21-18 的单倍体，仅发酵木糖时，木糖消耗率进一步提高了 10%。然而，它在发酵混合糖时不如其二倍体亲本。Z4X-21-18在对多重抑制剂含量高的全预处理麦秆浆进行预糖化-同步糖化发酵（P-SSF）时，乙醇产量约为42 g/L，总糖产量为0.38 g/g。葡萄糖和木糖的摄取率分别高出 1.4 倍和 8.3 倍。H18s28 是 Z4X-21-18 的单倍体，仅发酵木糖时，木糖消耗率进一步提高了 10%。然而，它在发酵混合糖时不如其二倍体亲本。Z4X-21-18在预糖化-同步糖化发酵（P-SSF）过程中，对多抑制剂含量高的全麦秸秆预处理液进行预糖化-同步糖化发酵（P-SSF），Z4X-21-18产乙醇约42 g/L，总糖产量0.38 g/g。