BACKGROUND Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. RESULTS In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae, were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha. CONCLUSIONS The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

中文翻译：

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糖转运蛋白的工程设计，用于提高多形小形酵母高温酒精发酵过程中木糖的利用率。

背景技术木糖运输是木质纤维素生物质向乙醇转化的瓶颈之一。一旦葡萄糖耗尽，野生型利用木糖的酵母菌株就会消耗木糖，从而导致漫长的发酵过程以及从木质纤维素水解产物中释放出来的糖的总体缓慢而不完全的转化。因此，用于促进葡萄糖-木糖共消耗的内源转运蛋白的工程设计是从木质纤维素水解物中有效生产乙醇的重要前提。结果在这项研究中，以前曾用于酿酒酵母中低亲和力葡萄糖转运蛋白的几种工程方法，已成功地用于多形小形藻中较早鉴定出的转运蛋白Hxt1，以改善木糖的消耗（工程涉及在358位的天冬酰胺取代为丙氨酸，N端赖氨酸残基的取代预计是精氨酸残基的泛素化目标）。此外，酿酒酵母Hxt7和Gal2转运蛋白的修饰形式在多形酵母中表达时还导致木糖发酵得到改善。结论具有修饰的Hxt1的O.polymorpha菌株的特征在于同时利用葡萄糖和木糖，与野生型和亲本菌株相比，木糖的乙醇产量增加。当将工程化的Hxt1转运蛋白引入木糖时，已被构建为较早的先进乙醇生产商，所得菌株显示出木糖发酵过程中乙醇积累的进一步增加。异源酿酒酵母Gal2的过表达对糖的吸收率没有那么深刻的积极影响，而Hxt7的过表达对糖的消耗影响最小。