Laboratory of Genetics and Biotechnology, EMBRAPA Agroenergy, Parque Estação Biológica, PqEB - W3 Norte Final s/no, 70.770-901, Brasília, Brazil.
US DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Appl Microbiol Biotechnol. 2023 Feb;107(4):1143-1157. doi: 10.1007/s00253-023-12362-1. Epub 2023 Jan 10.
Lignocellulosic biomass is a renewable raw material for producing several high-value-added chemicals and fuels. In general, xylose and glucose are the major sugars in biomass hydrolysates, and their efficient utilization by microorganisms is critical for an economical production process. Yeasts capable of co-consuming mixed sugars might lead to higher yields and productivities in industrial fermentation processes. Herein, we performed adaptive evolution assays with two xylose-fermenting yeasts, Spathaspora passalidarum and Scheffersomyces stipitis, to obtain derived clones with improved capabilities of glucose and xylose co-consumption. Adapted strains were obtained after successive growth selection using xylose and the non-metabolized glucose analog 2-deoxy-D-glucose as a selective pressure. The co-fermentation capacity of evolved and parental strains was evaluated on xylose-glucose mixtures. Our results revealed an improved co-assimilation capability by the evolved strains; however, xylose and glucose consumption were observed at slower rates than the parental yeasts. Genome resequencing of the evolved strains revealed genes affected by non-synonymous variants that might be involved with the co-consumption phenotype, including the HXT2.4 gene that encodes a putative glucose transporter in Sp. passalidarum. Expression of this mutant HXT2.4 in Saccharomyces cerevisiae improved the cells' co-assimilation of glucose and xylose. Therefore, our results demonstrated the successful improvement of co-fermentation through evolutionary engineering and the identification of potential targets for further genetic engineering of different yeast strains. KEY POINTS: • Laboratory evolution assay was used to obtain improved sugar co-consumption of non-Saccharomyces strains. • Evolved Sp. passalidarum and Sc. stipitis were able to more efficiently co-ferment glucose and xylose. • A mutant Hxt2.4 permease, which co-transports xylose and glucose, was identified.
木质纤维素生物质是生产多种高附加值化学品和燃料的可再生原料。一般来说，木糖和葡萄糖是生物质水解物中的主要糖，微生物对它们的有效利用对于经济的生产过程至关重要。能够共消耗混合糖的酵母可能会导致工业发酵过程中更高的产量和生产力。在此，我们对两种能够发酵木糖的酵母 Spathaspora passalidarum 和 Scheffersomyces stipitis 进行了适应性进化实验，以获得具有改进的葡萄糖和木糖共消耗能力的衍生克隆。在使用木糖和非代谢葡萄糖类似物 2-脱氧-D-葡萄糖作为选择性压力进行连续生长选择后，获得了适应的菌株。对进化菌株和亲本菌株在木糖-葡萄糖混合物上的共发酵能力进行了评估。我们的结果表明，进化菌株具有改善的共同化能力；然而，与亲本酵母相比，木糖和葡萄糖的消耗速度较慢。进化菌株的基因组重测序揭示了受非同义变体影响的基因，这些基因可能与共消耗表型有关，包括编码 Sp. passalidarum 中假定葡萄糖转运蛋白的 HXT2.4 基因。该突变 HXT2.4 在酿酒酵母中的表达提高了细胞对葡萄糖和木糖的共同化能力。因此，我们的结果表明，通过进化工程成功地提高了共发酵能力，并确定了不同酵母菌株进一步遗传工程的潜在目标。 关键点： • 使用实验室进化实验获得了非酿酒酵母提高的糖共消耗能力。 • 进化后的 Sp. passalidarum 和 Sc. stipitis 能够更有效地共发酵葡萄糖和木糖。 • 鉴定出一种突变的 Hxt2.4 通透酶，可共转运木糖和葡萄糖。
