Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake

Biosci Biotechnol Biochem. 2004 Jan;68(1):206-14. doi: 10.1271/bbb.68.206.

Abstract

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.

MeSH terms

  • Alcoholic Beverages
  • Caproates / metabolism*
  • Cell Division / drug effects
  • Cell Division / genetics
  • Cerulenin / pharmacology
  • Cloning, Molecular / methods*
  • Drug Resistance, Microbial / genetics
  • Fatty Acid Synthases / genetics*
  • Fatty Acid Synthases / metabolism
  • Genetic Engineering / methods
  • Point Mutation*
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Caproates
  • Saccharomyces cerevisiae Proteins
  • Cerulenin
  • Fatty Acid Synthases
  • ethyl hexanoate