Cell periphery-related proteins as major genomic targets behind the adaptive evolution of an industrial Saccharomyces cerevisiae strain to combined heat and hydrolysate stress

Valeria Wallace-Salinas, Daniel P. Brink, Dag Ahrén, Marie F. Gorwa-Grauslund

Laboratory evolution is an important tool for developing robust yeast strains for bioethanol production since the biological basis behind combined tolerance requires complex alterations whose proper regulation is difficult to achieve by rational metabolic engineering. Previously, we reported on the evolved industrial Saccharomyces cerevisiae strain ISO12 that had acquired improved tolerance to grow and ferment in the presence of lignocellulose-derived inhibitors at high temperature (39 °C). In the current study, we used comparative genomics to uncover the extent of the genomic alterations that occurred during the evolution process and investigated possible associations between the mutations and the phenotypic traits in ISO12. Through whole-genome sequencing and variant calling we identified a high number of strain-unique SNPs and INDELs in both ISO12 and the parental strain Ethanol Red. The variants were predicted to have 760 non-synonymous effects in both strains combined and were significantly enriched in Gene Ontology terms related to cell periphery, membranes and cell wall. Eleven genes, including MTL1, FLO9/FLO11, and CYC3 were found to be under positive selection in ISO12. Additionally, the FLO genes exhibited changes in copy number, and the alterations to this gene family were correlated with experimental results of multicellularity and invasive growth in the adapted strain. An independent lipidomic analysis revealed further differences between the strains in the content of nine lipid species. Finally, ISO12 displayed improved viability in undiluted spruce hydrolysate that was unrelated to reduction of inhibitors and changes in cell wall integrity, as shown by HPLC and lyticase assays. Together, the results of the sequence comparison and the physiological characterisations indicate that cell-periphery proteins (e.g. extracellular sensors such as MTL1) and peripheral lipids/membranes are important evolutionary targets in the process of adaptation to the combined stresses. The capacity of ISO12 to develop complex colony formation also revealed multicellularity as a possible evolutionary strategy to improve competitiveness and tolerance to environmental stresses (also reflected by the FLO genes). Although a panel of altered genes with high relevance to the novel phenotype was detected, this study also demonstrates that the observed long-term molecular effects of thermal and inhibitor stress have polygenetic basis.