Mutagenesis, breeding, and characterization of sake yeast strains with low production of dimethyl trisulfide precursor.

Dimethyl trisulfide (DMTS) is one of the main components responsible for hineka, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5'-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.

Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS).

Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called "hineka". Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Δmeu1, Δmri1, and Δmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Δmri1 and Δmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Δspe2 sake than in Δmri1 or Δmde1 sake, the DMTS producing potential of Δspe2 sake was as low as that of Δmri1 or Δmde1 sake. Sake brewing tests using BY4743 Δspe2 Δmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Δspe2 or Δmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake.