The bio3 mutation in sake yeast leads to changes in organic acid profiles and ester levels but not ethanol production.

Biotin is an essential coenzyme that is bound to carboxylases and participates in fatty acid synthesis. The fact that sake yeast exhibit biotin prototrophy while almost all other Saccharomyces cerevisiae strains exhibit biotin auxotrophy, implies that biotin prototrophy is an important factor in sake brewing. In this study, we inserted a stop codon into the biotin biosynthetic BIO3 gene (cording for 7,8-diamino-pelargonic acid aminotransferase) of a haploid sake yeast strain using the marker-removable plasmid pAUR135 and investigated the fermentation profile of the resulting bio3 mutant. Ethanol production was not altered when the bio3 mutant was cultured in Yeast Malt (YM) medium containing 10% glucose at 15 °C and 30 °C. Interestingly, ethanol production was also not changed during the sake brewing process. On the other hand, the levels of organic acids in the bio3 mutant were altered after culturing in YM medium and during sake brewing. In addition, ethyl hexanoate and isoamyl acetate levels decreased in the bio3 mutant during sake brewing. Metabolome analysis revealed that the decreased levels of fatty acids in the bio3 mutant were attributed to the decreased levels of ethyl hexanoate. Further, the transcription level of genes related to the synthesis of ethyl hexanoate and isoamyl acetate were significantly reduced. The findings indicated that although the decrease in biotin biosynthesis did not affect ethanol production, it did affect the synthesis of components such as organic acids and aromatic compounds. Biotin biosynthesis ability is thus a key factor in sake brewing.