Characteristic features of the unique house sake yeast strain Saccharomyces cerevisiae Km67 used for industrial sake brewing.

For several decades, almost all sake has been brewed with sake yeast Saccharomyces cerevisiae Kyokai no. 7 (K7) group strains. Although the widespread use of these strains has contributed to sake quality improvement, it may have lessened the diversity of sake gustatory properties brought about by house sake yeast (indigenous yeast of sake brewery). Sake yeast S. cerevisiae strain Km67 derives from the house yeast strain of Kiku-masamune Sake Brewing Co., Ltd., and it has been playing a central role in industrial sake brewing for decades. By using DNA sequencing, we revealed that strain Km67 does not possess specific loss-of-function mutations of stress response-related genes, which are characteristic of K7 group strains. Km67 had higher stress tolerance than K7 group strains likely because of the more efficient function of the stress response and heat shock elements in this strain. Sensory evaluation and taste sensor analysis demonstrated that sake brewed with Km67 had characteristically thicker body than sake brewed with K7 group strains. Chemical analysis suggested that unique sensory properties of the sake brewed with Km67 were due to high citramalic acid concentration. Taken together, these results revealed that strain Km67 differs from K7 group strains by genetic background and confers unique chemical composition and taste qualities upon sake it generates. It is expected that sake quality and gustatory properties will be diversified by utilizing house yeast such as strain Km67.

Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio.

Sake yeast produces a fruity flavor known as ginjo-ko-which is mainly attributable to ethyl caproate and isoamyl acetate-during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio from a cerulenin-resistant mutant derived from the hia1 strain, which shows enhanced isoamyl acetate productivity. The hec2 mutant had the homozygous FAS2 mutation Gly1250Ser, which is known to confer high ethyl caproate productivity. When the homozygous FAS2 mutation Gly1250Ser was introduced into strain hia1, ethyl caproate productivity was increased but neither this nor intracellular caproic acid content approached the levels observed in the hec2 mutant, indicating that a novel mutation was responsible for the high ethyl caproate productivity. We also found that the expression of EEB1 encoding acyl-coenzyme A:ethanol O-acyltransferase (AEATase) and enzymatic activity were increased in the hec2 mutant. These results suggest that the upregulation of EEB1 expression and AEATase activity may also have contributed to the enhancement of ethyl caproate synthesis from ethanol and caproyl-CoA. Our findings are useful for the brewing of sake with improved flavor due to high levels of isoamyl acetate and ethyl caproate.

Isolation and characterization of sake yeast mutants with enhanced isoamyl acetate productivity.

Isoamyl acetate is an important flavor compound in sake. However, production of isoamyl acetate by Saccharomyces cerevisiae is significantly reduced during sake brewing with rice that has a high polishing ratio, because unsaturated fatty acids derived from the outer layer of rice repress the expression of ATF1, which encodes an alcohol acetyl transferase. Yeast mutants capable of relieving this repression would allow the brewing of rice with high polishing ratios, improving the diversity of taste and flavor of sake. Atf1p is also believed to contribute to biological membrane homeostasis. We isolated four yeast mutants (hia1, hia2, hia4, and hia6) that have high isoamyl acetate productivity and are resistant to aureobasidin A, an inhibitor of sphingolipid biosynthesis. The isoamyl acetate content of sake brewed with the hia1 mutant was 2.6 times higher than that of the parental strain. ATF1 was expressed constitutively in the hia1 mutant during brewing and remained derepressed upon the addition of unsaturated fatty acids. Whole-genome sequence analysis of the hia mutants revealed a homozygous nonsense mutation (Ser706*) in MGA2 in all four mutants. Mga2p, an endoplasmic reticulum (ER) membrane protein, regulates ATF1 transcription. The expression of ATF1 was elevated in BY4743 Δmga2 cells complemented with MGA2 (Ser706*), and this was not completely inhibited by the addition of unsaturated fatty acids. These results indicate that a nonsense mutation in MGA2 induces high levels of isoamyl acetate production in S. cerevisiae. This finding has applications for brewing sake with high levels of isoamyl acetate.