Evaluation of adaptive mutations to etanol stress in yeasts

Abstract

A critical factor in alcoholic fermentations is the inhibition of yeasts cells by the product ethanol. For this reason, high alcoholic tolerance is a major requirement for yeast strains used in industry. In a previous project, we conducted ethanol adaptive evolution experiments in which we propagated the yeast Schizosaccharomyces japonicus for about 2700 generations on increasing amounts of alcohol (up to 10% v/v). In another protocol, we submitted four Saccharomyces cerevisiae PE-2 populations to about 70-80 shocks of 19% to 30% (v/v) ethanol. Genome sequencing of ethanol-adapted strains from both experiments revealed about 306 mutations. In the present project, we propose to study the evolved populations obtained in our previous experiments, and to analyze individually the most important mutations found by whole genome sequencing of both S. cerevisiae and S. japonicus. To this end, we will establish a series of comparative analyses to access ethanol tolerance in evolved yeast strains. They include cell viability assays after ethanol shocks, spot essays with or without ethanol, cell-wall strength analysis, metabolites' profile, controlled fermentations and, especially, competition assays (progenitor versus evolved) under the evolution experiment conditions. In a second moment, we will individually transform key mutant alleles into the parental genome to study the adaptive contribution of each respective mutation to the ethanol tolerance phenotype. Finally, the knowledge gathered will enable rational genetic engineering of S. cerevisiae PE-2 for enhanced ethanol tolerance. (AU)