Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ

Abstract Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae . To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase δ is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase δ, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1 , which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.