Sterol-targeted laboratory evolution allows the isolation of thermotolerant and respiratory-competent clones of the industrial yeast Saccharomyces cerevisiae

Evidence suggests that sterol content and composition play an important role in the ability of yeast cells to face high temperatures. Nevertheless, our knowledge of the exact mechanisms operating is still scarce, which makes the rational engineering of this industrial-relevant trait difficult. Here, we have used a fluconazole (FCNZ)-driven experimental evolution approach with the idea of inducing changes in the sterol biosynthesis pathway linked to high temperature tolerance. The evolution experiment rendered a FCNZ-resistant population of a previously selected bakers yeast strain, from which six isolates with increased thermotolerance were rescued. Initial characterization of evolved clones grouped them into two sets, switching between respiratory competence and deficiency. This late was connected to mtDNA loss, an event that appears to induce FCNZ and heat tolerance. Genome sequencing and ploidy-level analysis of all strains revealed aneuploidies, CNVs, and SNPs, which could contribute to phenotypic heterogeneity. In particular, fragment amplifications of Chr IV and XIV, which harbour dosage-sensitive genes, and specific SNPs in thermotolerance genes (AVT3, SFP1 and RNT1), could be on the basis of the phenotype of respiratory-competent evolved clones. In addition, all the evolved clones showed changes in their profiles of ergosterol biosynthesis intermediates, which again were different in respiratory-competent and defective strains. Hence, our experimental evolution allowed us to generate fully competent industrial strains with better performance at high temperatures, and identify new determinants of fluconazole and heat tolerance.