Efficient breeding of a non-urea-producing sake yeast from a wild Saccharomyces cerevisiae: a case study of a strain isolated from the Sado Gold Mine, Japan

栗林, 喬; Jumpei Tanaka; Masamichi Sugawara; Keigo Sato; Yoshihito Nabekura; Toshio Aoki

doi:10.51094/jxiv.3374

##article.authors##

栗林, 喬新潟県醸造試験場 https://orcid.org/0000-0002-0719-6005 https://researchmap.jp/takashi-kuribayashi
Jumpei Tanaka Niigata Prefectural Sake Research Institute
Masamichi Sugawara Niigata Prefectural Sake Research Institute
Keigo Sato Niigata Prefectural Sake Research Institute
Yoshihito Nabekura Niigata Prefectural Sake Research Institute
Toshio Aoki Niigata Prefectural Sake Research Institute

DOI:

https://doi.org/10.51094/jxiv.3374

キーワード:

alcoholic beverage、 arginase-deficient mutation、 FAS2、 sake making、 mating‑based yeast breeding

抄録

Herein, we developed a simple and rapid breeding technique to improve a wild, homothallic Saccharomyces cerevisiae strain (Sado8) isolated from plant litter at the UNESCO World Cultural Heritage Site of the Sado Gold Mine (Niigata, Japan). We enabled direct mating-based breeding of the wild Sado8 isolate without introducing additional auxotrophic markers by crossing it with a sake yeast haploid partner carrying an arginase-deficient mutation and the FAS2-G1250S mutation, and by employing a selective medium compatible with these markers. Using this approach, we bred the Sado8-derived strain LNY2, which exhibited high ethanol fermentation performance, high ethyl caproate production, and no detectable urea formation. Industrial-scale fermentation tests demonstrated that LNY2 consistently produces high-quality and safe sake. Furthermore, the arginase gene mutation retained in LNY2 provides an effective genetic marker for yeast strain authentication and traceability in sake production.

利益相反に関する開示

The authors declare that they have no conflict of interest.

ダウンロード *前日までの集計結果を表示します

ダウンロード実績データは、公開の翌日以降に作成されます。

引用文献

Akada, R., Matsuo, K., Aritomi, K., & Nishizawa, Y. (1999). Construction of recombinant sake yeast containing a dominant FAS2 mutation without extraneous sequences by a two-step gene replacement protocol. Journal of Bioscience and Bioengineering, 87(1), 43–48. https://doi.org/10.1016/S1389-1723(99)80006-1

Aoki, T. (2024). Sake research in Niigata Prefecture and the characteristics of Japanese sake. Seibutsu Kogaku Kaishi, 102(10), 509–511. https://doi.org/10.34565/seibutsukogaku.102.10_509

Coulombe, J. J., & Favreau, L. (1963). A new simple semimicro method for colorimetric determination of urea. Clinical Chemistry, 9(1), 102–108. https://doi.org/10.1093/clinchem/9.1.102

Fukuda, N., & Takeuchi, M. (2024). Removal of undesirable genes using yeast backcrossing. Journal of Bioscience and Bioengineering, 138(5), 369–374. https://doi.org/10.1016/j.jbiosc.2024.07.015

Hatakeyama, A., Watanabe, Y., Arimoto, K., Hara, T., & Joh, T. (2020). Genotypic analysis of the FDC1 polymorphism that inhibits ferulic acid decarboxylation activity in industrial yeasts. Journal of the Brewing Society of Japan, 115(4), 249–254. https://doi.org/10.6013/jbrewsocjapan.115.249

Huxley, C., Green, E. D., & Dunham, I. (1990). Rapid assessment of S. cerevisiae mating type by PCR. Trends in Genetics: TIG, 6(8), 236. https://doi.org/10.1016/0168-9525(90)90190-H

Ichikawa, E., Hosokawa, N., Hata, Y., Abe, Y., Suginami, K., & Imayasu, S. (1991). Breeding of a sake yeast with improved ethyl caproate productivity. Agricultural and Biological Chemistry, 55(8), 2153–2154. https://doi.org/10.1271/bbb1961.55.2153

Inokoshi, J., Tomoda, H., Hashimoto, H., Watanabe, A., Takeshima, H., & Ōmura, S. (1994). Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene. Molecular and General Genetics: MGG, 244(1), 90–96. https://doi.org/10.1007/BF00280191

Johansson, P., Wiltschi, B., Kumari, P., Kessler, B., Vonrhein, C., Vonck, J., Oesterhelt, D., & Grininger, M. (2008). Inhibition of the fungal fatty acid synthase type I multienzyme complex. Proceedings of the National Academy of Sciences of the United States of America, 105(35), 12803–12808. https://doi.org/10.1073/pnas.0805827105

Kitagaki, H., & Kitamoto, K. (2013). Breeding research on sake yeasts in Japan: History, recent technological advances, and future perspectives. Annual Review of Food Science and Technology, 4(1), 215–235. https://doi.org/10.1146/annurev-food-030212-182545

Kitamoto, K., Miyazaki, K., Yamaoka, H., Gomi, K., & Kumagai, C. (1992). Isolation of sake yeasts producing no urea without mutagenesis treatment. Journal of the Brewing Society of Japan, 87(8), 598–601. https://doi.org/10.6013/jbrewsocjapan1988.87.598

Kitamoto, K., Oda-Miyazaki, K., Gomi, K., & Kumagai, C. (1993). Mutant isolation of non-urea producing sake yeast by positive selection. Journal of Fermentation and Bioengineering, 75(5), 359–363. https://doi.org/10.1016/0922-338X(93)90134-T

Kuribayashi, T., Sato, K., Joh, T., & Kaneoke, M. (2017). Breeding of a non-urea-producing sake yeast carrying a FAS2 mutation. Mycoscience, 58(4), 302–306. https://doi.org/10.1016/j.myc.2017.02.005

Kuribayashi, T., Sawaguchi, F., Satone, H., Tanaka, J., Sugawara, M., Sato, K., Nabekura, Y., & Aoki, T. (2025). Fermentation characteristics and brewing potential of kuratsuki sake yeasts isolated from the Niigata Prefecture, Japan. Jxiv. https://doi.org/10.51094/jxiv.1308

Kuribayashi, T., Tamura, H., & Watanabe, K. (2015). Isolation of a non-urea-producing sake yeast strain carrying a discriminable molecular marker: And its application to industrial brewing. Journal of the Brewing Society of Japan, 110(4), 182–188. https://doi.org/10.6013/jbrewsocjapan.110.182

Kuribayashi, T., Tsukada, M., Asahi, N., Kai, S. I., Abe, K. I., Kaneoke, M., Oguma, T., Kinebuchi, J., Shigeno, T., Sugiyama, T., & Kasai, D. (2024). Screening using loop-mediated isothermal amplification (LAMP) assay and breeding of a Saccharomyces cerevisiae strain isolated from Muramatsu Park, Japan, for sake brewing. Mycoscience, 65(4), 187–190. https://doi.org/10.47371/mycosci.2024.04.002

Li, M., & Jia, W. (2024). Formation and hazard of ethyl carbamate and construction of genetically engineered Saccharomyces cerevisiae strains in huangjiu (Chinese grain wine). Comprehensive Reviews in Food Science and Food Safety, 23(2), e13321. https://doi.org/10.1111/1541-4337.13321

Mao, X., Yue, S. J., Xu, D. Q., Fu, R. J., Han, J. Z., Zhou, H. M., & Tang, Y. P. (2023). Research progress on flavor and quality of Chinese rice wine in the brewing process. ACS Omega, 8(36), 32311–32330. https://doi.org/10.1021/acsomega.3c04732

McClure, A. W., Jacobs, K. C., Zyla, T. R., & Lew, D. J. (2018). Mating in wild yeast: Delayed interest in sex after spore germination. Molecular Biology of the Cell, 29(26), 3119–3127. https://doi.org/10.1091/mbc.E18-08-0528

Mitsui, S., Yamamoto, K., Ito, A., Ieda, A., Kondo, T., Sugiyama, N., Akao, T., Yoshimura, A., Sakakibara, Y., Funai, H., Haramoto, N., Shimizu, M., & Kato, M. (2021). Use of synchrotron light as a mutagen for breeding non-urea producing Aichi sake yeast. Journal of the Brewing Society of Japan, 116(10), 703–712. https://doi.org/10.6013/jbrewsocjapan.116.703

Mortimer, R. K. (2000). Evolution and variation of the yeast (Saccharomyces) genome. Genome Research, 10(4), 403–409. https://doi.org/10.1101/gr.10.4.403

Namba, Y., Obata, T., Kayashima, S., Yamasaki, Y., Murakami, M., & Shimoda, T. (1978). Conditions of small scale sake brewing. Journal of the Brewing Society of Japan, 73(4), 295–300. https://doi.org/10.6013/jbrewsocjapan1915.73.295

Nikulin, J., Vidgren, V., Krogerus, K., Magalhães, F., Valkeemäki, S., Kangas-Heiska, T., & Gibson, B. (2020). Brewing potential of the wild yeast species Saccharomyces paradoxus. European Food Research and Technology, 246(11), 2283–2297. https://doi.org/10.1007/s00217-020-03572-2

Peter, J., De Chiara, M., Friedrich, A., Yue, J.-X., Pflieger, D., Bergström, A., Sigwalt, A., Barre, B., Freel, K., Llored, A., Cruaud, C., Labadie, K., Aury, J.-M., Istace, B., Lebrigand, K., Barbry, P., Engelen, S., Lemainque, A., Wincker, P., … Schacherer, J. (2018). Genome evolution across 1,011 Saccharomyces cerevisiae isolates. Nature, 556(7701), 339–344. https://doi.org/10.1038/s41586-018-0030-5

Tanaka, J., Sugawara, M., Kuribayashi, T., Sato, K., Nabekura, Y., & Aoki, T. (2024). Study on improvement of Niigata sake yeast -Isolation of a no-urea-producing sake yeast with high ethyl caproate productivity-. Quest for Traditinal Food, 50, 55–60. https://doi.org/10.69331/questtraditionalfood.50.0_55

Tonouchi, A., Moriyama, Y., Aoyama, Y., & Toki, H. (2016). Application of the yeast Saccharomyces cerevisiae isolated from the Shirakami Mountains. Journal of the Brewing Society of Japan, 111(7), 437–444. https://doi.org/10.6013/jbrewsocjapan.111.437

Washizu, S., & Yamazaki, Y. (1974). Ginjo sake making by Toji of Niigata Prefecture. Journal of the Brewing Society of Japan, 69(8), 495–498. https://doi.org/10.6013/jbrewsocjapan1915.69.495

Weber, J. V., & Sharypov, V. I. (2009). Ethyl carbamate in foods and beverages–a review. In E. Lichtfouse (Ed.), Climate Change, Intercropping, Pest Control and Beneficial microorganisms (pp. 429–452). Springer Netherlands. https://doi.org/10.1007/978-90-481-2716-0_15

Yoshida, K. (2006). Kyoukai sake Yeast. Journal of the Brewing Society of Japan, 101(12), 910–922. https://doi.org/10.6013/jbrewsocjapan1988.101.910

Yoshiuchi, K., Watanabe, M., & Nishimura, A. (2000). Breeding of a non-urea producing sake yeast with killer character using a kar1-1 mutant as a killer donor. Journal of Industrial Microbiology and Biotechnology, 24(3), 203–209. https://doi.org/10.1038/sj.jim.2900797

Yoshizawa, K. (1999). Sake: Production and flavor. Food Reviews International, 15(1), 83–107. https://doi.org/10.1080/87559129909541178