Improving ethanol tolerance of Saccharomyces cerevisiae through adaptive laboratory evolution using high ethanol concentrations as a selective pressure

Saccharomyces cerevisiae is a microorganism of remarkable biotechnological interest, owing to its involvement in bioethanol production, as well as in beverages and bread production. High ethanol concentration during alcoholic fermentation constitutes a major stress for yeast cells, leading to decreased fermentation rate and reduced ethanol production. Herein we describe an innovative Adaptive Laboratory Evolution (ALE) strategy for the acquisition of S. cerevisiae populations more tolerant to ethanol toxicity. In this strategy, a weak selective pressure environment (encouraging diversity) is alternated by a strong selective pressure environment (eliminating low-tolerant clones). Two different parental strains, named S. cerevisiae CFB and S. cerevisiae BLR, were used and the resulting evolved populations obtained after 100 generations of evolution were proven capable of surviving at 23% v/v ethanol for 1 h and at 25% v/v ethanol for 2 h, respectively. Two evolved populations originated from S. cerevisiae CFB and S. cerevisiae BLR after 150 and 200 generations of evolution respectively, exhibited higher fermentation rates than their parental strains in synthetic broth with 200 g/l glucose, completing the fermentation 166 h and 130 h earlier, respectively. The evolved populations derived after 100 generations of evolution produced 94.77 and 98.67 g/l ethanol, for S. cerevisiae CFB and S. cerevisiae BLR, respectively, in comparison with 84.91 and 78.09 g/l of their parental strains. Contrary, the maximum specific growth rate (μmax) was improved solely in S. cerevisiae BLR, reaching the value of 0.25 1/h after 100 generations of evolution in comparison with 0.12 1/h of the parental strain. It is concluded that the fermentation ability of the two S. cerevisiae strains was significantly improved through evolution using ethanol as a selective factor.