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J Biosci Bioeng. 2010 Dec;110(6):675-8. doi: 10.1016/j.jbiosc.2010.07.007. Epub 2010 Aug 12.

The construction and application of diploid sake yeast with a homozygous mutation in the FAS2 gene.

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Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto 612-8385, Japan.


In Japanese sake brewing, cerulenin-resistant sake yeasts produce elevated levels of ethyl caproate, an important flavor component. The FAS2 mutation FAS2-1250S of Saccharomyces cerevisiae generates a cerulenin-resistant phenotype. This mutation is dominant, and, in general, cerulenin-resistant diploid sake yeast strains carry this mutation heterozygously. Here we constructed diploid sake yeast with a homozygous mutation of FAS2 using the high-efficiency loss of heterozygosity method. The homozygous mutants grew more slowly in YPD medium than did the wild-type and heterozygous mutants, and they produced more ethyl caproate during sake brewing. In addition, although both the wild-type and heterozygous mutant were sensitive to 4 mg/l cerulenin, the homozygous mutant was resistant to more than 4 mg/l cerulenin. Next, we obtained a homozygous mutant of FAS2 without inducing genetic modification. After cultivating the heterozygous FAS2 mutant K-1801 in YPD, homozygous mutants were selected on medium containing high concentrations of cerulenin. Non-genetically modified yeast with a homozygous mutation of FAS2 produced 2.2-fold more ethyl caproate than did heterozygous yeast. Moreover, high-quality Japanese sake with a very rich flavor could be brewed using yeast containing a homozygous mutation in the FAS2 gene.

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