Advertisement

Applied Microbiology and Biotechnology

, Volume 66, Issue 1, pp 108–114 | Cite as

Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells

  • Shingo Izawa
  • Machiko Sato
  • Kumio Yokoigawa
  • Yoshiharu InoueEmail author
Applied Microbial and Cell Physiology

Abstract

Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1Δ gcy1Δ gre3Δ ypr1Δ) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.

Keywords

Trehalose Freeze Tolerance Freeze Storage Freeze Stress Glycerol Dehydrogenase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We are grateful to Dr. W. Bandlow (gcy1Δ∷LEU2) and Dr. S. Harashima for providing yeast strains and plasmids. We also thank Mr. T. Tanaka and Mr. T. Suzuki for their technical support in the construction of yeast mutants. This study was supported by the Iijima Memorial Foundation for the Promotion of Food Science and Technology and Bio-oriented Technology Research Advancement Institution (BRAIN).

References

  1. Albertyn J, Hohmann S, Thevelein J, Prior BA (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14:4135–4144PubMedGoogle Scholar
  2. Ansell R, Granath K, Hohmann S, Thevelein J, Adler L (1997) The two isozymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 16:2179–2187PubMedGoogle Scholar
  3. Coutinho C, Bernardes E, Felix D, Panek AD (1988) Trehalose as cryoprotectant for preservation of yeast strains. J Biotechnol 7:23–32CrossRefGoogle Scholar
  4. Eriksson P, André L, Ansell R, Blomberg A, Adler L (1995) Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. Mol Microbiol 17:95–107PubMedGoogle Scholar
  5. Hino A, Mihara K, Nakashima K, Takano H (1990) Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts. Appl Environ Microbiol 56:1386–1391PubMedGoogle Scholar
  6. Hirasawa R, Yokoigawa K (2001) Leavening ability of baker’s yeast exposed to hyperosmotic media. FEMS Microbiol Lett 194:159–162CrossRefPubMedGoogle Scholar
  7. Hirasawa R, Yokoigawa K, Isobe Y, Kawai H (2001) Improving the freeze tolerance of bakers’ yeast by loading with trehalose. Biosci Biotechnol Biochem 65:522–526PubMedGoogle Scholar
  8. Holst B, Lunde C, Lages F, Oliveira R, Lucas C, Kielland-Brandt MC (2000) GUP1 and its close homologue GUP2, encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae. Mol Microbiol 37:108–124CrossRefPubMedGoogle Scholar
  9. Izawa S, Maeda K, Sugiyama K, Mano J, Inoue Y, Kimura A (1999) Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae. J Biol Chem 274:28459–28465CrossRefPubMedGoogle Scholar
  10. Kaul SC, Obuchi K, Iwahashi H, Komatsu Y (1992) Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae cells: induction of a 33 kDa protein and protection against freezing injury. Cell Mol Biol 38:135–143PubMedGoogle Scholar
  11. Kim S, Huh WK, Lee BH, Kang SO (1998) d-Arabinose dehydrogenase and its gene from Saccharomyces cerevisiae. Biochim Biophys Acta 1429:29–39CrossRefPubMedGoogle Scholar
  12. Kitada K, Yamaguchi E, Arisawa M (1995) Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation. Gene 165:203–206CrossRefPubMedGoogle Scholar
  13. Kuhn A, van Zyl C, van Tonder A, Prior BA (1995) Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae. Appl Environ Microbiol 61:1580–1585Google Scholar
  14. Larsson K, Ansell R, Eriksson P, Adler L (1993) A gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae. Mol Microbiol 10:1101–1111PubMedGoogle Scholar
  15. Lewis JG, Learmonth RP, Watson K (1993) Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae. Appl Environ Microbiol 59:1065–1071PubMedGoogle Scholar
  16. Lewis JG, Learmonth RP, Watson K (1995) Induction of heat, freezing and salt tolerance by heat and salt shock in Saccharomyces cerevisiae. Microbiology 141:687–694PubMedGoogle Scholar
  17. Lorenz MC, Muir RS, Lim E, McElver J, Weber SC, Heitman J (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113–117CrossRefPubMedGoogle Scholar
  18. Luyten K, Albertyn J, Skibbe W, Prior BA, Ramos J, Thevelein JM, Hohmann S (1995) Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and it is inactive under osmotic stress. EMBO J 14:1360–1371PubMedGoogle Scholar
  19. Michnick S, Roustan JL, Remize F, Barre P, Dequin S (1997) Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol-3-phosphate dehydrogenase. Yeast 13:783–793CrossRefPubMedGoogle Scholar
  20. Murakami Y, Yokoigawa K, Kawai F, Kawai H (1996) Lipid composition of commercial baker’s yeast having different freeze-tolerance in frozen dough. Biosci Biotechnol Biochem 60:1874–1876PubMedGoogle Scholar
  21. Myers DK, Joseph VM, Pehm S, Galvagno M, Attfield PV (1998) Loading of Saccharomyces cerevisiae with glycerol leads to enhanced fermentation in sweet bread doughs. Food Microbiol 15:51–58CrossRefGoogle Scholar
  22. Nakagawa S, Ouchi K (1994) Construction from a single parent of baker’s yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs. Appl Environ Microbiol 60:3499–3502PubMedGoogle Scholar
  23. Nakamura K, Kondo S, Kawai Y, Nakajima N, Ohno A (1997) Amino acid sequence and characterization of aldo-keto reductase from baker’s yeast. Biosci Biotechnol Biochem 61:375–377PubMedGoogle Scholar
  24. Nissen T, Hamann CW, Kielland-Brandt MC, Nielsen J, Villadsen J (2000) Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis. Yeast 16:463–474CrossRefPubMedGoogle Scholar
  25. Oechsner U, Magdolen V, Bandlow W (1988) A nuclear yeast gene (GCY) encodes a polypeptide with high homology to a vertebrate eye lens protein. FEBS Lett 238:123–128CrossRefPubMedGoogle Scholar
  26. Park J-I, Grant CM, Attfield PV, Dawes IW (1997) The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway. Appl Environ Microbiol 63:3813–3824Google Scholar
  27. Pavlik P, Simon M, Schuster T, Ruis H (1993) The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization. Curr Genet 24:21–25Google Scholar
  28. Påhlman AK, Granath K, Ansell R, Hohmann S, Adler L (2001) The yeast glycerol-3-phosphatases Gpp1 and Gpp2 are required for glycerol biosynthesis and differentially involved in the cellular response to osmotic, anaerobic, and oxidative stress. J Biol Chem 276:3555–3563CrossRefPubMedGoogle Scholar
  29. Remize F, Roustan JL, Sablayrolles JM, Barre P, Dequin S (1999) Glycerol overproduction by engineered Saccharomyces cerevisiae wine yeast strains leads to substantial changes in byproduct formation and to a stimulation of fermentation rate in stationary phase. Appl Environ Microbiol 65:143–149PubMedGoogle Scholar
  30. Remize F, Barnavon L, Dequin S (2001) Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. Metab Eng 3:301–312CrossRefPubMedGoogle Scholar
  31. Schuller C, Brewster JL, Alexander MR, Gustin MC, Ruis H (1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of Saccharomyces cerevisiae CTT1 gene. EMBO J 13:4382–4389PubMedGoogle Scholar
  32. Shima J, Hino A, Yamada-Iyo C, Suzuki Y, Nakajima R, Watanabe H, Mori K, Takano H (1999) Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial baker’s yeast. Appl Environ Microbiol 65:2841–2846PubMedGoogle Scholar
  33. Shima J, Sakata-Tsuda Y, Suzuki Y, Nakajima R, Watanabe H, Kawamoto S, Takano H (2003) Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker’s yeast Saccharomyces cerevisiae. Appl Environ Microbiol 69:715–718CrossRefPubMedGoogle Scholar
  34. Siderius M, Wuytswinkel OV, Reijenga KA, Kelders M, Mager WH (2000) The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature. Mol Microbiol 36:1381–1390CrossRefPubMedGoogle Scholar
  35. Sutherland FCW, Lages F, Lucas C, Luyten K, Albertyn J, Hohmann S, Prior BA, Kilian SG (1997) Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae. J Bacteriol 179:7790–7795PubMedGoogle Scholar
  36. Takagi H, Iwamoto F, Nakamori S (1997) Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants. Appl Microbiol Biotechnol 47:405–411CrossRefPubMedGoogle Scholar
  37. Tamas MJ, Luyten K, Sutherland F, Hernandez A, Albertyn J, Valadi H, Li H, Prior B, Kilian S, Ramos J, Gustafsson L, Thevelein J, Hohmann S (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol Microbiol 31:1087–1094PubMedGoogle Scholar
  38. Tanghe A, Dijck PV, Dumortier F, Teunissen A, Hohmann S, Thevelein JM (2002) Aquaporin expression correlates with freeze tolerance in baker’s yeast, and overexpression improves freeze tolerance in industrial strains. 68:5981–5989Google Scholar
  39. Toh T-H, Kayingo G, van der Merwe MJ, Kilian SG, Hallsworth JE, Hohmann S, Prior BA (2001) Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae. FEMS Yeast Res 1:205–211CrossRefPubMedGoogle Scholar
  40. Träff KL, Jönsson LJ, Hahn-Hägerdal B (2002) Putative xylose and arabinose reductases in Saccharomyces cerevisiae. Yeast 19:1233–1241CrossRefPubMedGoogle Scholar
  41. Vries RP de, Flitter SJ, van de Vondervoort PJ, Chaveroche MK, Fontaine T, Fillinger S, Ruijter GJ, d’Enfert C, Visser J (2003) Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans. Mol Microbiol 49:131–141CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Shingo Izawa
    • 1
  • Machiko Sato
    • 2
  • Kumio Yokoigawa
    • 2
  • Yoshiharu Inoue
    • 1
    Email author
  1. 1.Laboratory of Molecular Microbiology, Graduate School of AgricultureKyoto UniversityUjiJapan
  2. 2.Department of Food Science and NutritionNara Women’s UniversityNaraJapan

Personalised recommendations