清酒酵母高発酵性変異株の開発とその機作の解明
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High ethanol concentrations in sake mash, which characteristically reach over 20%, occasionally cause stuck fermentation resulting in low-quality sake. Adequate fermentative activity is thus one of the most important features required of a sake yeast. Mutants resistant to clotrimazole (CTZ) were isolated from the sake yeasts Kyokai no. 701 and no. 1001. More than half of them fermented sake mash faster and produced higher amounts of alcohol than their parental strains. In industrial-scale sake fermentation, a CTZ-resistant mutant could reduce the fermentation period by 1-2 days. CTZ-resistant mutants from a non-urea forming sake yeast and a high isoamyl acetate producing strain showed improved fermentative activity while retaining their parental advantages for sake making. CTZ-resistant mutants also exhibit pleiotropic drug resistance (PDR); they are resistant to a wide range of cytotoxic compounds unrelated to CTZ. Series of gain-of-function missense mutations in PDR1 and PDR3, which encode transcription factors, are known to increase the expression of ABC transporters that extrude various kinds of drugs out of the cell, leading to PDR. In this study, two PDR1 and three PDR3 mutant alleles were cloned from five independent CTZ-resistant mutants. These alleles contain different missense mutations -PDR1-H176 (L309S), PDR1-2 (M308I), PDR3-C21 (L950S), PDR3-C231 (G948D), and PDR3-11 (G957D)- that are sufficient to improve fermentative activity, in addition to conferring PDR. In a CTZ-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YORJ, markedly increased, while the cellular ATP contents drastically decreased. Disruption of PDR5 or SNQ2 but not YOR1 in CTZ21 lowered the fermentative activity to that of HL69 and restored the cellular ATP contents to a medium level between those of CTZ21 and HL69. These results demonstrated that the ATP level reduced by overexpression of PDR5 and SNQ2 in CTZ21 contributes to a certain extent to its improved fermentative activity.
- 社団法人日本生物工学会の論文
- 2002-02-25
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