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Direct mating between diploid sake strains of Saccharomyces cerevisiae

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Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.

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  1. Acuña G, Würgler FE, Sengstag C (1994) Reciprocal mitotic recombination is the predominant mechanism for the loss of a heterozygous gene in Saccharomyces cerevisiae. Environ Mol Mutagen 24:307–316

  2. Akada R (2002) Genetically modified industrial yeast ready for application. J Biosci Bioeng 94:536–544

  3. 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 replacement protocol. J Biosci Bioeng 87:43–48

  4. Aritomi K, Hirosawa I, Hoshida H, Shiigi M, Nishizawa Y, Kashiwagi S, Akada R (2004) Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake. Biosci Biotechnol Biochem 68:206–214

  5. Bell PJL, Deere D, Shen J, Chapman B, Bissinger PH, Attfield PV, Veal DA (1998) A flow cytometric method for rapid selection of novel industrial yeast hybrids. Appl Environ Microbiol 64:1669–1672

  6. Benitez T, Gasent-Ramirez JM, Castrejon F, Codon AC (1996) Development of new strains for the food industry. Biotechnol Prog 12:149–163

  7. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

  8. Brewing Society of Japan (1993) Annotation of the official analytical methods of the National Tax Administration Agency of Japan, 4th edn. The Brewing Society of Japan, Tokyo, pp 13–24

  9. Dequin S (2001) The potential of genetic engineering for improving brewing, wine-making and baking yeasts. Appl Microbiol Biotechnol 56:577–588

  10. Guijo S, Mauricio JC, Salmon JM, Ortega JM (1997) Determination of the relative ploidy in different Saccharomyces cerevisiae strains used for fermentation and ‘flor’ film ageing of dry sherry-type wines. Yeast 13:101–117

  11. Gunge N (1966) Breeding of bakers' yeast-determination of the ploidy and an attempt to improve practical properties. Japan J Genet 41:203–214

  12. Gunge N, Nakatomi Y (1972) Genetic mechanisms of rare mating of Saccharomyces cerevisiae heterozygous for mating type. Genetics 70:41–58

  13. Hammond JRM (1995) Genetically-modified brewing yeasts for the 21st century. Progress to date. Yeast 11:1613–1627

  14. Hashimoto S, Ogura M, Aritomi K, Hoshida H, Nishizawa Y, Akada R (2005) Isolation of auxotrophic mutants of diploid industrial yeast strains after UV mutagenesis. Appl Environ Microbiol 71:312–319

  15. Higgins VJ, Bell PJL, Dawes IW, Attfield PV (2001) Generation of a novel Saccharomyces cerevisiae strain that exhibits strong maltose utilization and hyperosmotic resistance using nonrecombinant techniques. Appl Environ Microbiol 67:4346–4348

  16. Hiraoka M, Watanabe K, Umezu K, Maki H (2000) Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells. Genetics 156:1531–1548

  17. Hirosawa I, Aritomi K, Hoshida H, Kashiwagi S, Nishizawa Y, Akada R (2004) Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol. Appl Microbiol Biotechnol 65:68–73

  18. Javadekar VS, SivaRaman H, Gokhale DV (1995) Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion. J Ind Microbiol 15:94–102

  19. Kashiwagi S (2002) Merchandizing of sake made from ‘Yamaguchi Sakura Yeast’. J Brew Soc Jpn 97:2–6 (in Japanese)

  20. Kishimoto M (1994) Fermentation characteristics of hybrids between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae. J Ferment Bioeng 77:432–435

  21. Lima N, Moreira C, Teixeira JA, Mota M (1995) Introduction of flocculation into industrial yeast, Saccharomyces cerevisiae saké, by protoplast fusion. Microbios 81:187–197

  22. Lindegren CC, Lindegren G (1943) Selecting, inbreeding, recombining, and hybridizing commercial yeasts. J Bacteriol 46:405–419

  23. Loray MA, Spencer JFT, Spencer DM, de Figueroa LIC (1995) Hybrids obtained by protoplast fusion with a salt-tolerant yeast. J Ind Microbiol 14:508–513

  24. Maráz A (2002) From yeast genetics to biotechnology. Acta Microbiol Immunol Hung 49:483–491

  25. Nakazawa N, Ashikari T, Goto N, Amachi T, Nakajima R, Harashima S, Oshima Y (1992) Partial restoration of sporulation defect in sake yeasts, Kyokai no. 7 and no. 9, by increased dosage of the IME1 gene. J Ferment Bioeng 73:265–270

  26. Nakazawa N, Hashimoto H, Harashima S, Oshima Y (1993) Use of the PDR4 gene as a dominant selective marker in combination with cerulenin for prototrophic strains in Saccharomyces cerevisiae. J Ferment Bioeng 76:60–63

  27. Nakazawa N, Tsuchihara K, Hattori T, Akita K, Harashima S, Oshima Y (1994) A method for direct selection of mating-competent clones from mating-incompetent industrial strains of Saccharomyces cerevisiae. J Ferment Bioeng 78:6–11

  28. Nakazawa N, Okawa K, Sato T, Enei H, Harashima S (1999) Mass mating method in combination with G418- and aureobasidin A-resistance markers for efficient selection of hybrids from homothallic strains in Saccharomyces cerevisiae. J Biosci Bioeng 88:468–471

  29. Pretorius IS (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16:675–729

  30. Pretorius IS, Bauer FF (2002) Meeting the consumer challenge through genetically customized wine–yeast strains. Trends Biotech 20:426–432

  31. Ramírez M, Peréz F, Regodón JA (1998) A simple and reliable method for hybridization of homothallic wine strains of Saccharomyces cerevisiae. Appl Environ Microbiol 64:5039–5041

  32. Romano P, Soli MG, Suzzi G, Grazia L, Zambonelli C (1985) Improvement of a wine Saccharomyces cerevisiae strain by a breeding program. Appl Environ Microbiol 50:1064–1067

  33. Scheinbach S (1983) Protoplast fusion as a means of producing new industrial yeast strains. Biotechnol Adv 1:289–300

  34. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York

  35. Shinohara T, Saito K, Yanagida F, Goto S (1994) Selection and hybridization of wine yeasts for improved winemaking properties: fermentation rate and aroma productivity. J Ferment Bioeng 77:428–431

  36. Shinohara T, Mamiya S, Yanagida F (1997) Introduction of flocculation property into wine yeasts (Saccharomyces cerevisiae) by hybridization. J Ferment Bioeng 83:96–101

  37. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

  38. Spencer JFT, Spencer DM (1977) Hybridization of non-sporulating and weakly sporulating strains of brewer's and distiller's yeast. J Inst Brew 83:287–289

  39. Suizu T, Tsutsumi H, Kawado A, Imayasu S, Inose T, Kimura A, Murata K (1994) Induction of yeast sporulation by lysine-related compounds and glutathione in nutrition-rich conditions. J Ferment Bioeng 77:568–571

  40. Suizu T, Tsutsumi H, Kawado A, Murata K, Suginami K, Imayasu S (1996) Methods for sporulation of industrially used sake yeasts. J Ferment Bioeng 81:93–97

  41. Tamai Y, Tanaka K, Kaneko Y, Harashima S (2001) HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette. Appl Microbiol Biotechnol 55:333–340

  42. Teunissen A, Dumortier F, Gorwa M, Bauer J, Tanghe A, Loïez A, Smet P, Van Dijck P, Thevelein JM (2002) Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs. Appl Environ Microbiol 68:4780–4787

  43. Tsuboi M, Takahashi T (1988) Genetic analysis of the non-sporulating phenotype of brewer's yeasts. J Ferment Technol 66:605–613

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We would like to thank Dr. Harumi Suzuki in Yamaguchi University Medical School, Japan, for his help with flow cytometric analysis, and Mr. Takahiro Nagayama in Nagayama Honke Shuzo, Japan, for his help in making sake. This work was supported in part by the RSP program of Japan Science and Technology Corporation.

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Correspondence to Rinji Akada.

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Hashimoto, S., Aritomi, K., Minohara, T. et al. Direct mating between diploid sake strains of Saccharomyces cerevisiae . Appl Microbiol Biotechnol 69, 689–696 (2006).

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  • Industrial Strain
  • Isoamyl Acetate
  • Auxotrophic Mutation
  • Yeast Peptone Dextrose
  • Opposite Mating Type