ABSTRACT
An evolution and resequencing strategy was used to research the genetic basis of Saccharomyces cerevisiae BR20 (with 18 vol% ethanol tolerance) and the evolved strain F23 (with 25 vol% ethanol tolerance). Whole-genome sequencing and RNA sequencing (RNA-seq) indicated that the enhanced ethanol tolerance under 10 vol% ethanol could be attributed to amino acid metabolism, whereas 18 vol% ethanol tolerance was due to fatty acid metabolism. Ultrastructural analysis indicated that F23 exhibited better membrane integrity than did BR20 under ethanol stress. At low concentrations (<5 vol%), the partition of ethanol into the membrane increased the membrane fluidity, which had little effect on cell growth. However, the toxic effects of medium and high ethanol concentrations (5 to 20 vol%) tended to decrease the membrane fluidity. Under high ethanol stress (>10 vol%), the highly tolerant strain was able to maintain a relatively constant fluidity by increasing the content of unsaturated fatty acid (UFA), whereas less-tolerant strains show a continuous decrease in fluidity and UFA content. OLE1, which was identified as the only gene with a differential single-nucleotide polymorphism (SNP) mutation site related to fatty acid metabolism, was significantly changed in response to ethanol. The role of OLE1 in membrane fluidity was positively validated in its overexpressed transformants. Therefore, OLE1 lowered the rate of decline in membrane fluidity and thus enabled the yeast to better fight the deleterious effects of ethanol.
IMPORTANCE Yeasts with superior ethanol tolerance are desirable for winemakers and wine industries. In our previous work, strain F23 was evolved with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Therefore, exploring the genomic variations and ethanol tolerance mechanism of strain F23 could contribute to an understanding of its effect on the flavor characteristics in the resulting Chinese rice wine. The cellular membrane plays a vital role in the ethanol tolerance of yeasts; however, how the membrane is regulated to fight the toxic effect of ethanol remains to be elucidated. This study suggests that the membrane fluidity is variably regulated by OLE1 to offset the disruptive effect of ethanol. Current work will help develop more ethanol-tolerant yeast strains for wine industries and contribute to a deep understanding of its high flavor-producing ability.
Complete genome sequence and analysis of the industrial Saccharomyces cerevisiae strain N85 used in Chinese rice wine production
