Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv, 79005, Ukraine.
Department of Microbiology and Biotechnology, University of Rzeszow, Zelwerowicza 4, Rzeszow, 35-601, Poland.
Microb Cell Fact. 2020 Apr 25;19(1):96. doi: 10.1186/s12934-020-01354-9.
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.
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.
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.
木糖运输是将木质纤维素生物质转化为乙醇的瓶颈之一。木糖利用酵母的野生型菌株在葡萄糖耗尽后才消耗木糖，导致发酵过程漫长，木质纤维素水解物中释放的糖的整体转化缓慢且不完全。因此，工程化内源性转运蛋白以促进葡萄糖-木糖共消耗是从木质纤维素水解物中高效生产乙醇的重要前提。
在这项研究中，以前用于酿酒酵母低亲和力葡萄糖转运蛋白的几种工程方法，成功地应用于先前鉴定的 Ogataea polymorpha 中的转运蛋白 Hxt1，以提高木糖消耗（工程涉及将第 358 位的天冬酰胺取代为丙氨酸，以及取代预测为泛素化目标的 N 端赖氨酸残基为精氨酸残基）。此外，当在 O. polymorpha 中表达时，改良的 S. cerevisiae Hxt7 和 Gal2 转运蛋白的版本也导致木糖发酵得到改善。
与野生型和亲本菌株相比，具有改良 Hxt1 的 O. polymorpha 菌株的特征是同时利用葡萄糖和木糖，而不是利用木糖提高乙醇产量。当将工程化的 Hxt1 转运蛋白引入之前构建的从木糖生产乙醇的先进工程菌株中时，在木糖发酵过程中，该菌株表现出更高的乙醇积累。异源 S. cerevisiae Gal2 的过表达对糖摄取率的影响较小，而 Hxt7 的过表达对糖消耗的影响最小。
