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Improving lactic acid (LA) tolerance is important for cost-effective microbial production of LA under acidic fermentation conditions. Previously, we generated LA-tolerant D-LA-producing  S. cerevisiae  strain JHY5310 by laboratory adaptive evolution of JHY5210. In this study, we performed whole genome sequencing of JHY5310, identifying four loss-of-function mutations in  GSF2 ,  SYN8 ,  STM1 , and  SIF2  genes, which are responsible for the LA tolerance of JHY5310. Among the mutations, a nonsense mutation in  GSF2  was identified as the major contributor to the improved LA tolerance and LA production in JHY5310. Deletion of  GSF2  in the parental strain JHY5210 significantly improved glucose uptake and D-LA production levels, while derepressing glucose-repressed genes including genes involved in the respiratory pathway. Therefore, more efficient generation of ATP and NAD +  via respiration might rescue the growth defects of the LA-producing strain, where ATP depletion through extensive export of lactate and proton is one of major reasons for the impaired growth. Accordingly, alleviation of glucose repression by deleting  MIG1  or  HXK2  in JHY5210 also improved D-LA production.  GSF2  deletion could be applied to various bioprocesses where increasing biomass yield or respiratory flux is desirable.

GSF2 deletion increases lactic acid production by alleviating glucose repression in Saccharomyces cerevisiae