Identification of Traits to Improve Co-Assimilation of Glucose and Xylose by Adaptive Evolution of Spathaspora Passalidarum and Scheffersomyces Stipitis Yeasts.

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.