Breeding of a non-urea-producing sake yeast carrying a FAS2 mutation

We isolated non-urea-producing sake yeasts from a FAS2-G3748A (G1250S) mutant using established positive-screening medium (containing canavanine, arginine and ornithine; CAO medium) after selection of FAS2 mutants with cerulenin. Varying levels of ethyl caproate production were observed due to spontaneous allelic recombination of the FAS2 heterozygotes on the CAO medium. The resulting double mutants exhibited favorable fermentation properties for sake brewing.