Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

中文翻译：

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进化工程酿酒酵母菌株在D-木糖上高效，对D-葡萄糖不敏感的生长。

优化酿酒酵母中的D-木糖消耗量对于生产具有成本效益的纤维素生物乙醇至关重要。进化工程方法被用于提高内源嵌合Hxt36己糖转运蛋白中携带D-木糖特异性N367I突变的木糖发酵酿酒酵母菌株中的D-木糖消耗。该菌株携带四倍的己糖激酶缺失，阻止了葡萄糖的利用，并允许在高D-葡萄糖浓度下选择D-木糖上提高的生长速率。进化工程导致D-葡萄糖不敏感的D-木糖生长和消耗，这可以归因于HXT36和HXT7基因融合产生的新型嵌合Hxt37 N367I转运蛋白对葡萄糖不敏感的D-木糖的摄取，并下调了一组介导葡萄糖敏感性木糖转运的Hxt转运蛋白。RNA测序显示HXT1和HXT2的下调，以及HXT7的缺失，导致所有质膜转运蛋白基因的表达降低21％。形态分析表明，进化菌株的细胞大小增加，细胞表面积相应增加，这可能是由于基因组重复造成的。与顺序消耗D-葡萄糖和D-的菌株相比，消耗D-木糖的菌株DS71054-evo6与消耗D-葡萄糖的CEN.PK113-7D菌株的混合菌株发酵可降低残留糖浓度并提高乙醇产量。木糖。RNA测序显示HXT1和HXT2的下调，以及HXT7的缺失，导致所有质膜转运蛋白基因的表达降低21％。形态学分析显示进化株的细胞大小增加和细胞表面积相应增加，这可能归因于基因组重复。与顺序消耗D-葡萄糖和D-的菌株相比，消耗D-木糖的菌株DS71054-evo6与消耗D-葡萄糖的CEN.PK113-7D菌株的混合菌株发酵可降低残留糖浓度并提高乙醇产量。木糖。RNA测序显示HXT1和HXT2的下调，以及HXT7的缺失，导致所有质膜转运蛋白基因的表达降低21％。形态分析表明，进化菌株的细胞大小增加，细胞表面积相应增加，这可能是由于基因组重复造成的。与顺序消耗D-葡萄糖和D-的菌株相比，消耗D-木糖的菌株DS71054-evo6与消耗D-葡萄糖的CEN.PK113-7D菌株的混合菌株发酵可降低残留糖浓度并提高乙醇产量。木糖。形态分析表明，进化菌株的细胞大小增加，细胞表面积相应增加，这可能是由于基因组重复造成的。与顺序消耗D-葡萄糖和D-的菌株相比，消耗D-木糖的菌株DS71054-evo6与消耗D-葡萄糖的CEN.PK113-7D菌株的混合菌株发酵可降低残留糖浓度并提高乙醇产量。木糖。形态学分析显示进化株的细胞大小增加和细胞表面积相应增加，这可能归因于基因组重复。与顺序消耗D-葡萄糖和D-的菌株相比，消耗D-木糖的菌株DS71054-evo6与消耗D-葡萄糖的CEN.PK113-7D菌株的混合菌株发酵可降低残留糖浓度并提高乙醇产量。木糖。