Removal of undesirable genes using yeast backcrossing.

4-Vinylguaiacol (4-VG) is one of the causative compounds for the phenolic odor characteristics of brewed liquor. In the case of Japanese sake brewing, 4-VG is formed through the decarboxylation of ferulic acid produced by rice koji enzymes from steamed rice. PAD1 (phenylacrylic acid decarborxylase gene) and FDC1 (ferulic acid decarboxylase gene) genes are essential for decarboxylation of ferulic acid in Saccharomyces cerevisiae and the single polymorphisms of both genes show a relationship with ferulic acid decarboxylation ability. While most of the Kyokai yeasts distributed by the Brewing Society of Japan have homozygous non-function fdc1 alleles, many newly isolated natural yeasts for local specialities carry the wild-type FDC1 gene. In our previous research, we found that a crossbreed strain lost a significant amount of chromosomal DNA as it underwent meiosis-like adaptation. Here, we established a breeding approach to exclude undesirable gene alleles (such as the wild-type FDC1 genes) from yeast strains of interest by backcrossing using Kyokai yeasts. Homozygous fdc1 crossbreeds were generated through the three rounds of crossing procedures, and we confirmed the reduction of 4-VG in the culture supernatant of the homozygous fdc1 hybrid strain compared to the parental strain. Importantly, this approach does not include growth selection for the mutation of interest (the fdc1 mutant in the current case). Using various yeast strains generated throughout human history, it may become possible to design and build any yeast strains according to the purpose.