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

  • Applied Microbial and Cell Physiology
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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.

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References

  • 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 14:4135–4144

    CAS  PubMed  Google Scholar 

  • 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 16:2179–2187

    CAS  PubMed  Google Scholar 

  • Coutinho C, Bernardes E, Felix D, Panek AD (1988) Trehalose as cryoprotectant for preservation of yeast strains. J Biotechnol 7:23–32

    Article  CAS  Google Scholar 

  • 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 17:95–107

    CAS  PubMed  Google Scholar 

  • Hino A, Mihara K, Nakashima K, Takano H (1990) Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts. Appl Environ Microbiol 56:1386–1391

    CAS  PubMed  Google Scholar 

  • Hirasawa R, Yokoigawa K (2001) Leavening ability of baker’s yeast exposed to hyperosmotic media. FEMS Microbiol Lett 194:159–162

    Article  CAS  PubMed  Google Scholar 

  • Hirasawa R, Yokoigawa K, Isobe Y, Kawai H (2001) Improving the freeze tolerance of bakers’ yeast by loading with trehalose. Biosci Biotechnol Biochem 65:522–526

    CAS  PubMed  Google Scholar 

  • 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 37:108–124

    Article  CAS  PubMed  Google Scholar 

  • 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 274:28459–28465

    Article  CAS  PubMed  Google Scholar 

  • 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 38:135–143

    CAS  PubMed  Google Scholar 

  • Kim S, Huh WK, Lee BH, Kang SO (1998) d-Arabinose dehydrogenase and its gene from Saccharomyces cerevisiae. Biochim Biophys Acta 1429:29–39

    Article  CAS  PubMed  Google Scholar 

  • 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 165:203–206

    Article  CAS  PubMed  Google Scholar 

  • 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 61:1580–1585

    Google Scholar 

  • 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 10:1101–1111

    CAS  PubMed  Google Scholar 

  • Lewis JG, Learmonth RP, Watson K (1993) Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae. Appl Environ Microbiol 59:1065–1071

    CAS  PubMed  Google Scholar 

  • Lewis JG, Learmonth RP, Watson K (1995) Induction of heat, freezing and salt tolerance by heat and salt shock in Saccharomyces cerevisiae. Microbiology 141:687–694

    CAS  PubMed  Google Scholar 

  • Lorenz MC, Muir RS, Lim E, McElver J, Weber SC, Heitman J (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113–117

    Article  CAS  PubMed  Google Scholar 

  • 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 14:1360–1371

    CAS  PubMed  Google Scholar 

  • 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 13:783–793

    Article  CAS  PubMed  Google Scholar 

  • 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 60:1874–1876

    CAS  PubMed  Google Scholar 

  • 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 15:51–58

    Article  CAS  Google Scholar 

  • 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 60:3499–3502

    CAS  PubMed  Google Scholar 

  • 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 61:375–377

    CAS  PubMed  Google Scholar 

  • 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 16:463–474

    Article  CAS  PubMed  Google Scholar 

  • 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 238:123–128

    Article  CAS  PubMed  Google Scholar 

  • 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 63:3813–3824

    Google Scholar 

  • Pavlik P, Simon M, Schuster T, Ruis H (1993) The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization. Curr Genet 24:21–25

    Google Scholar 

  • 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 276:3555–3563

    Article  PubMed  Google Scholar 

  • 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 65:143–149

    PubMed  Google Scholar 

  • 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 3:301–312

    Article  CAS  PubMed  Google Scholar 

  • 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 13:4382–4389

    PubMed  Google Scholar 

  • 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 65:2841–2846

    CAS  PubMed  Google Scholar 

  • 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 69:715–718

    Article  CAS  PubMed  Google Scholar 

  • 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 36:1381–1390

    Article  CAS  PubMed  Google Scholar 

  • 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 179:7790–7795

    CAS  PubMed  Google Scholar 

  • Takagi H, Iwamoto F, Nakamori S (1997) Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants. Appl Microbiol Biotechnol 47:405–411

    Article  CAS  PubMed  Google Scholar 

  • 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 31:1087–1094

    PubMed  Google Scholar 

  • 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. 68:5981–5989

  • 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 1:205–211

    Article  CAS  PubMed  Google Scholar 

  • Träff KL, Jönsson LJ, Hahn-Hägerdal B (2002) Putative xylose and arabinose reductases in Saccharomyces cerevisiae. Yeast 19:1233–1241

    Article  PubMed  Google Scholar 

  • 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 49:131–141

    Article  PubMed  Google Scholar 

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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).

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Correspondence to Yoshiharu Inoue.

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Izawa, S., Sato, M., Yokoigawa, K. et al. Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells. Appl Microbiol Biotechnol 66, 108–114 (2004). https://doi.org/10.1007/s00253-004-1624-4

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