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Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.

Construction of uracil and tryptophan auxotrophic mutants from sake yeasts by disruption of URA3 and TRP1 genes.