Improvement of 2,3-butanediol tolerance in Saccharomyces cerevisiae by using a novel mutagenesis strategy

Abstract

Although the yeast Saccharomyces cerevisiae has been used to produce various bio-based chemicals, including solvents and organic acids, most of these products inhibit yeast growth at high concentrations. In general, it is difficult to rationally improve stress tolerance in yeast by modifying specific genes, because many of the genes involved in stress response remain unidentified. Previous studies have reported that various forms of stress tolerance in yeast were improved by introducing random mutations, such as DNA point mutations and DNA structural mutations. In this study, we developed a novel mutagenesis strategy that allows for the simultaneous performance of these two types of mutagenesis to construct a yeast variant with high 2,3-butanediol (2,3-BDO) tolerance. The mutations were simultaneously introduced into S. cerevisiae YPH499, accompanied by a stepwise increase in the concentration of 2,3-BDO. The resulting mutant YPH499/pol3δ/BD_392 showed 4.9-fold higher cell concentrations than the parental strain after 96 h cultivation in medium containing 175 g/L 2,3-BDO. Afterwards, we carried out transcriptome analysis to characterize the 2,3-BDO-tolerant strain. Gene ontology enrichment analysis with RNA sequence data revealed an increase in expression levels of genes related to amino acid metabolic processes. Therefore, we hypothesize that the yeast acquired high 2,3-BDO tolerance by amino acid function. Our research provides a novel mutagenesis strategy that achieves efficient modification of the genome for improving tolerance to various types of stressors.

Keywords: 2,3-Butanediol; CRISPR; DNA polymerase δ; Random mutagenesis; Saccharomyces cerevisiae; Stress tolerance.