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One of the most outstanding features of Saccharomyces cerevisiae sake yeast strains is the high fermentation rate in the sake mash. The molecular mechanism underlying this brewing property has been remained unrevealed, however. Our genome-wide comparative analyses of sake yeast and laboratory yeast strains led to identification of mutations responsible for the high fermentation ability of sake yeast.To identify differences between the sake and laboratory yeast strains that contribute to the superior brewing properties of sake yeast, we compared the gene expression profiles of both types of yeast in fermenting sake mash. Sake yeast showed remarkably reduced expression through Msn2/4p and Hsf1p, representative stress-responsive transcription factors in Saccharomyces cerevisiae. In contrast, yeast stress responses and resultant stress tolerance are generally considered to be important characteristics for effective ethanol fermentation. Therefore, we examined stress tolerance of sake yeast cells in detail, and found that sake yeast exhibited lower survival rates than laboratory yeast under heat shock or ethanol stress. These results demonstrate that sake yeast is defective in stress response in spite of the high fermentation ability.To elucidate the genetic variations responsible for the defective stress response of sake yeast, we searched for mutations in the genes related to Msn2/4p and Hsf1p by utilizing the whole genome sequence data of a representative sake yeast strain Kyokai No. 7. Consequently, we found novel mutations in Kyokai No. 7, including (i) a single nucleotide substitution that causes deletion of the zinc-finger motifs located at the carboxyl terminus of Msn4p, which abrogates the functions of Msn4p as a transcriptional activator; (ii) a single nucleotide insertion that leads to a frameshift of the RIM15 gene, encoding a protein kinase acting upstream of Msn2/4p; and (iii) loss of the entire PPT1 gene locus, whose product is related to regulatory dephosphorylation of Hsf1p. It is noteworthy that all these mutations were distributed only in the sake strains genetically close to Kyokai No. 7. Furthermore, we confirmed that each of these mutations significantly improved the fermentation rate of laboratory strains. Altogether, these sake yeast-specific mutations originate both the stress-sensitive phenotype and the high fermentation rate of sake yeast strains.These results provide novel insights into yeast stress responses as major impediments of effective ethanol fermentation, which are widely applicable to the development and improvement of advanced yeast strains in the bakery, brewing, and biofuel industries.