Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake.
Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.
Aritomi
Kazuo
K
Yamaguchi Prefectural Industrial Technology Institute, Ube, Japan.
Hirosawa
Isao
I
Hoshida
Hisashi
H
Shiigi
Mikio
M
Nishizawa
Yoshinori
Y
Kashiwagi
Susumu
S
Akada
Rinji
R
D000434:Alcoholic Beverages; D002208:Caproates / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002569:Cerulenin / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D004352:Drug Resistance, Microbial / Q000235:genetics; D064429:Fatty Acid Synthases / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D002208:Caproates; D029701:Saccharomyces cerevisiae Proteins; D002569:Cerulenin; D064429:Fatty Acid Synthases; C079237:ethyl hexanoate