Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae

Abstract

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

“…It may be envisaged that, in early lager-brewing processes, unstandardized mashing processes generated wort with a higher maltotriose content, which would have allowed for continued yeast growth during the maltotriose consumption phase. During serial transfer on sugar mixtures, the selective advantage of consuming a specific sugar from a mixture correlates with the number of generations on that sugar during each cycle (Papapetridis et al 2018; Wisselink et al 2009). Such conditions would therefore have conferred a significant selective advantage to a maltotriose-assimilating S. cerevisiae x S. eubayanus hybrid, especially if, similar to current ale yeasts, the S. cerevisiae parent was unable to ferment maltotriose.…”

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)

“…These rationally designed genetic modifications, combined with alternating adaptive evolution in xylose and lignocellulosic hydrolysates, resulted in a final strain, with excellent xylose fermentation that had an enhanced resistance to inhibitors [110]. Glucose/xylose co-fermentation was activated after HXK2 deletion and introduction of a GAL83 G673T allele which provided 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain [138].…”

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

“…The replicon of the pRS416 vector used in this work is derived from yeast plasmid 2µ, and plasmid copy numbers can vary from culture to culture [34]. Furthermore, in laboratory evolution of S. cerevisiae for growth on xylose, cells may acquire diverse chromosomal mutations that cause improved growth [35,36]. To prove that in our case the selected mutations in the PirXI structural gene caused improved growth on xylose, the mutations were reconstructed by site-directed mutagenesis in the original PirXI gene and cloned in a vector that was not subjected to previous selection.…”