The Identification of a Novel Gene and Its Effect on Stress Tolerance in Saccharomyces cerevisiae Chinese Rice Wine Strain

Abstract: They identified a putative gene g5170 from Saccharomyces cerevisiae (S. cerevisiae)， a Chinese rice wine strain D2， and characterized its effects on stress tolerance and fermentation performance. A protein encoded by g5170 contains an amidase domain， and was over-expressed in growth culture in YPD medium. A knockout strain D2Δg5170 is constructed by using Cre/loxP system. A disruption cassette of g5170 with a short flanking homologous regions to g5170 and kanr as selectable marker was amplified and transformed into S. cerevisiae through LiAc/SS carrier DNA/PEG method. Positive transformants were screened on G418 plates and verified by PCR. The kanr marker was removed by transforming plasmid pSH65 into the positive transformants and inducing expression of Cre recombinase by galactose. The procedure was repeated to obtain the knockout strain D2Δg5170， in which two g5170 copies were completely deleted. Compared to a wild type strain， mutant D2Δg5170 showed no significant alterations of tolerance to heat stress and osmotic stress， but exhibited significant lower tolerance to ethanol stress. In contrast， over-expression of g5170 in S. cerevisiae resulted in enhanced tolerance to ethanol stress. Simulation of rice wine fermentation showed that the growth rate， ethanol production and other parameters of the mutant were similar to that of the wild strain. These results suggested that g5170 was associated with ethanol stress response， which might impact fermentation performance of S. cerevisiae in rice wine production.

CSTR: 32200.14.cjcb.2018.08.0010