Mutant isolation of non-urea producing sake yeast by positive selection

Urea is reported to be a main precursor in wine and sake (Japanese rice wine) of ethyl carbamate (ECA), a suspected carcinogen. We constructed an arginase-deficient mutant (Δcar1/Δcar1) from a diploid sake yeast, Kyokai no. 9, using a gene disruption method (Kitamoto, K. et al., Appl. Environ. Microbiol., 57, 301, 1991). The car1 mutant thus constructed enabled us to brew sake containing no urea or ECA. In spite of their superior characteristics, industrial use of mutants for sake brewing has so far been difficult because guidelines for recombinant DNA utilization in the food and beverages industry have not yet been established. Using the genetically engineered car1 mutant, we have developed a new medium for the positive selection of car1 mutants. Many arginase-deficient mutants could be easily isolated from not only a laboratory haploid strain (X2180-1A), but also sake yeasts (Kyokai no. 9 and Kyokai no 10) and wine yeasts (Geisenheim 74 and Eperney). Sake with no urea could be brewed using the car1 mutants, and no ECA was detected in the resulting sake even after heat treatment (hi-ire) and storage.