Generation of an evolved Saccharomyces cerevisiae strain with a high freeze tolerance and an improved ability to grow on glycerol

  • Annamaria Merico
  • Enrico Ragni
  • Silvia Galafassi
  • Laura Popolo
  • Concetta Compagno
Original Paper


Glycerol is a residue generated during biodiesel production and represents around 10% of the total product output. Biodiesel production is currently having a significant impact on glycerol price, leading to an increased interest in the use of glycerol as a cheap substrate for fermentation processes. We have analysed the growth kinetics of two wild-type strains of Saccharomyces cerevisiae grown on synthetic media containing glycerol as the sole carbon and energy source. Both strains were initially unable to grow when cultivated under these conditions, and an unusually long lag phase was necessary prior to the appearance of slow-growing cells. Following the application of an “evolutionary engineering” approach, we obtained S. cerevisiae strains with an improved ability to grow on glycerol. We report here the isolation of an evolved strain that exhibits a reduction of the lag phase, a threefold increase of the specific growth rate and a higher glycerol consumption rate compared to wild-type strains. The evolved strain has retained its fermentative activity, producing ethanol at the same rate and yield as the wild type. Interestingly, the yeast biomass obtained by cultivating the evolved strain on synthetic glycerol-based media also showed a high viability after prolonged storage at −20°C. The strategy adopted in our study could be easily applied to obtain S. cerevisiae strains with new industrially relevant traits, such as an improved ability to use cheap substrates and high resistance to freeze and thaw procedures.


Saccharomyces cerevisiae Glycerol metabolism Freeze tolerance Yeast production Fermentation 



This work was supported by Programma Università per la Ricerca (PUR) 2008 to C.C. A. Merico and E. Ragni were recipients of a type A contract from Università degli Studi di Milano.


  1. 1.
    Adler L, Blomberg A, Nilsson A (1985) Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii. J Bacteriol 162:300–306PubMedGoogle Scholar
  2. 2.
    André L, Hemming A, Adler L (1991) Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+). FEBS 286:13–17CrossRefGoogle Scholar
  3. 3.
    Blomberg A (2000) Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol Lett 182:1–8PubMedCrossRefGoogle Scholar
  4. 4.
    Easterling ER, French TW, Hernandez ML (2009) The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis. Bioresour Technol 100:356–361PubMedCrossRefGoogle Scholar
  5. 5.
    Ferreira C, van Voorst F, Martins A, Neves L, Oliveira R, Kielland-Brandt MC, Lucas C, Brandt A (2005) A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae. Mol Biol Cell 16:2068–2076PubMedCrossRefGoogle Scholar
  6. 6.
    Gancedo C, Llobell A, Ribas J-C, Luchi F (1986) Isolation and characterization of mutants from Schyzosaccharomyces pombe defective in glycerol catabolism. Eur J Biochem 159:171–174PubMedCrossRefGoogle Scholar
  7. 7.
    Gonzalez E, Fernandez MR, Larroy C, Sola L, Pericas MA, Pares X, Biosca JA (2000) Characterization of a (2R, 3R)-2, 3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060 W gene product. Disruption and induction of the gene. J Biol Chem 275:35876–35885PubMedCrossRefGoogle Scholar
  8. 8.
    Hofmann KH, Babel W (1980) Dihydroxyacetone kinase of methanol-assimilating yeast. I. Regulation of dihydroxyacetone kinase from Candida methylica in situ. Z Allg Mikrobiol 20:389–398PubMedCrossRefGoogle Scholar
  9. 9.
    Hohmann S (2009) Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae. FEBS Lett 583:4025–4029PubMedCrossRefGoogle Scholar
  10. 10.
    Izawa S, Sato M, Yokoigawa K, Inoue Y (2004) Intracellular glycerol influence resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells. Appl Microbiol Biotechnol 66:108–114PubMedCrossRefGoogle Scholar
  11. 11.
    Khan A, Bhide A, Gadre R (2009) Mannitol production from glycerol by resting cells of Candida magnoliae. Bioresour Technol 100:4911–4913PubMedCrossRefGoogle Scholar
  12. 12.
    Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijeken JP, Pronk JT (2005) Evolutionary engineering of mixed-sugar utilisation by a xylose-fermenting Saccharomyces cerevisiae. FEMS Yeast Res 5:925–934PubMedCrossRefGoogle Scholar
  13. 13.
    Larsson C, Påhlman I-L, Ansell R, Rigoulet M, Adler L, Gustafsson L (1998) The importance of glycerol-3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. Yeast 14:347–357PubMedCrossRefGoogle Scholar
  14. 14.
    Luttik MAH, Overkamp KM, Kötter P, de Vries S, van Dijken JP, Pronk JT (1998) The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH. J Biol Chem 273:24529–24534PubMedCrossRefGoogle Scholar
  15. 15.
    Luyten K, Albertyn J, Skibbe W, Prior B, Thevelein J, Hohmann S (1995) Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J 14:1360–1371PubMedGoogle Scholar
  16. 16.
    Makri A, Fakas S, Aggelis G (2010) Metabolic activity of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch. Bioresour Technol 101:2351–2358PubMedCrossRefGoogle Scholar
  17. 17.
    Nevoigt E (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 72:379–412PubMedCrossRefGoogle Scholar
  18. 18.
    Oliveira R, Lages F, Silva-Graça M, Lucas C (2003) Fps1p channel is the mediator of the major part of glycerol passive diffusion in Saccharomyces cerevisiae: artefacts and re-definitions. Biochim Biophys Acta 1613:57–71PubMedCrossRefGoogle Scholar
  19. 19.
    Overkamp KM, Bakker BM, Kötter P, van Tuijl A, de Vries S, van Dijken JP, Pronk JT (2000) In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria. J Bacteriol 182:2823–2830PubMedCrossRefGoogle Scholar
  20. 20.
    Påhlman I-L, Larsson C, Averét N, Bunoust O, Boubekeur S, Gustafsson L, Rigoulet M (2002) Kinetic regulation of the mitochondrial glycerol-3-phosphate deydrogenase by the external NADH deydrogenase in Saccharomyces cerevisiae. J Biol Chem 277:27991–27995PubMedCrossRefGoogle Scholar
  21. 21.
    Panadero J, Pallotti C, Rodriguez-Vargas S, Randez-Gil F, Prieto JA (2006) A downshift in temperature activates the high osmolarity glycerol (HOG) pathway which determines freeze tolerance in Saccharomyces cerevisiae. J Appl Chem 281:4638–4645Google Scholar
  22. 22.
    Parawira W (2009) Biotechnological production of biodiesel fuel using biocatalysed transesterification: a review. Crit Rev Biotechnol 29:82–93PubMedCrossRefGoogle Scholar
  23. 23.
    Pavlik P, Simon M, Schuster T, Ruis H (1993) The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization. Curr Genet 24:21–25PubMedCrossRefGoogle Scholar
  24. 24.
    Rigoulet M, Aguilaniu H, Averet N, Bunoust O, Camougrand N, Grandier-Vazeille X, Larsson C, Pahlman I-L, Manon S, Gustafsson L (2004) Organisation and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae. Mol Cell Biochem 256(257):73–81PubMedCrossRefGoogle Scholar
  25. 25.
    Rønnow B, Kielland-Brandt MC (1993) GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae. Yeast 9:1121–1130PubMedCrossRefGoogle Scholar
  26. 26.
    Sauer U (2001) Evolutionary engineering of industrially important microbial phenotypes. Adv Biochem Eng Biotechnol 73:129–169PubMedGoogle Scholar
  27. 27.
    Sherman F, Hicks J (1991) Micromanipulation and dissection of asci. In: Guthrie C, Fink G (eds) Methods in enzymology, vol 194. London, Academic Press, pp 21–37Google Scholar
  28. 28.
    Sprague GF, Cronan JE (1977) Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol metabolism. J Bacteriol 129:1335–1342PubMedGoogle Scholar
  29. 29.
    van der Klei IJ, van der Heide M, Baerends RJ, Rechinger KB, Nicolay K, Kiel JA, Veenhuis M (1998) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity. Curr Genet 34:1–11PubMedCrossRefGoogle Scholar
  30. 30.
    van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Rev 32:199–225Google Scholar
  31. 31.
    van Zyl PJ, Prior BA, Kilian SG (1991) Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress. Appl Microbiol Biotechnol 36:369–374CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  • Annamaria Merico
    • 1
  • Enrico Ragni
    • 1
  • Silvia Galafassi
    • 1
  • Laura Popolo
    • 1
  • Concetta Compagno
    • 1
  1. 1.Dipartimento di Scienze Biomolecolari e BiotecnologieUniversità degli Studi di MilanoMilanItaly

Personalised recommendations