Combinatorial gene overexpression and recessive mutant gene introduction in sake yeast.

Industrial yeast strains are generally diploid and are often defective in sporulation. Such strains are hence thought to be less tractable for manipulation by genetic engineering. To facilitate more reliable genetic manipulation of the diploid yeast Japanese sake, we constructed variants of this strain that were homozygous for a URA3 deletion, homozygous for either MATa or MATalpha, and homozygous for either the his3 or the lys4 mutation.

Direct mating between diploid sake strains of Saccharomyces cerevisiae.

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium.

Isolation of auxotrophic mutants of diploid industrial yeast strains after UV mutagenesis.

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His(-), Met(-), Lys(-), Trp(-), Leu(-), Arg(-), and Ura(-) auxotrophic mutants of five sake strains, Kyokai no.

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.