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 (ΔURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (ΔURA3) 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 (ΔURA3) 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 (ΔTRP1 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.
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