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

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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.

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Yeast strains and media

The sake yeast Saccharomyces cerevisiae strain Kyokai no. 701 (K701) was provided by the Brewing Society of Japan. Two additional haploid strains derived from K701 owned by the Nihonsakari Co., Ltd. (Nishinomiya, Japan), 9-1 (MATα) and #4 (MATa), were also used. The methionine-auxotrophic strain (MO-001, MATα) was obtained from 9-1 strain. MDR01 was produced by the disruption of the MRI1 gene of MO-001 strain.

YPD medium (1% yeast extract, 2% bacto-peptone, and 2% glucose) was used for yeast

Establishment of a screening method for MTA non-utilizing strains

The methionine-auxotrophic strain MO-001 was generated from the wild-type strain 9-1 by EMS mutation treatment, and was selected from strains that did not grow in the minimal culture medium, but grew in minimal culture medium containing methionine, and formed black or brown colonies due to the generation of lead sulfide in MLA medium, using the replica method. The medium conditions were examined using the MRI1 gene disrupted strain MDR01 obtained from MO-001 strain. First, acquisition was

Discussion

In this study, the MTA method was established as a screening method for MTA non-utilizing strains targeting genes involved in the production of DMTS-P1, a precursor of DMTS and the main component of hineka. The mutants were screened using the MTA method to obtain candidate strains. These candidate strains were confirmed to have mutations in the MDE1 or MRI1 gene, and the DMTS-P1 production ability was low, as expected. However, the fermentation abilities of the strains were poor, and therefore,

Acknowledgments

We wish to thank Dr. T. Goshima for technical suggestions on FACS analysis.

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Present address: Faculty of Food and Agricultural Sciences, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan.

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