Improving Ethanol Tolerance in Industrial Yeast Strain by a Combination of Mutation and Evolutionary Engineering

Tolerance to ethanol is a key characteristic of the yeast Saccharomyces cerevisiae. Any increase in ethanol tolerance in the industrial strains could lead to faster and more complete fermentations, and may also allow the production of more alcohol. Due to the complex nature of ethanol tolerance, it appears that a large increase in ethanol tolerance requires several changes in the yeast’s genome. Several approaches that rely on the effect of (random) variation generated by evolutionary engineering or mutagenesis have successfully yielded strains with increased ethanol tolerance.

In the present study, to improve the ethanol tolerance phenotype in the industrial Ethanol Red strain, the parent strain was mutated physically and chemically. The mutants were screened using 1-butanol containing medium. The primary parent and the mutants were evolved within 144 days with evolutionary engineering strategy, while ethanol production of the selected strains was investigated

According to the increase in the maximum growth rate, 8 strains were selected including parental strain and mutants, and the amounts of ethanol production of these strains were evaluated after evolutionary adaptation tests. Ethanol production of ER 103 and ER 106 which were mutated with EMS before the adaptive evolution test and then evolved at 11 and 9% v/v ethanol was improved from 103.44 ± 0.5 g/L to 112.45 ± 1 and 112.3 ± 0.9 g/L, respectively.

Due to the extensive capabilities of the evolutionary engineering method in creating capable strains in order to increase ethanol tolerance in industrial strains, the evolutionary engineering strategy was used. To increase the genetic diversity of the primary population, before starting the adaptive evolution experiments, mutation with ethyl methane sulfonate was used, which was more efficient than ultraviolet radiation in accelerating the evolution process to achieve the desired phenotype.