Isolation and Pilot-scale Evaluation of Isoamyl Alcohol-overproducing Mutants of Bottom-fermenting Brewing Yeasts

5,5,5-trffluoro-DL-leucine (TFL)-resistant bottom-fermenting yeast mutants were isolated and found to overproduce both isoamyl alcohol and isoamyl acetate. The TFL-resistant strains were found to harbor dominant mutations in either the S. cerevisiae (SC)-type or S. bayanus (SB)-type alleles of LEU4 encoding a-isopropylmalate synthase (alpha-IPM synthase). All TFL-resistant mutants were isolated as heterozygotes. When a single wild-type allele in the parent strain was replaced with a mutant LEU4 allele, overproduction of isoamyl alcohol and TFL resistance was observed, indicating that a single dominant mutation was responsible for both phenotypes. In addition, feedback inhibition of a-isopropylmalate synthase (alpha-IPM synthase) by L-leucine was found to be weaker in the mutant than in the parent. On a pilot-scale brewing experiment, the original mutants with the dominant LEU4 allele produced beer containing more isoamyl alcohol and isoamyl acetate than the parent strain.