Skip to main content
SpringerLink
Log in

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ12-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Hodgkin MA, Sudbery PE, Kerry-Williams S, Goodey A (1990) Secretion of human serum albumin from Hansenula polymorpha. Yeast 6S:435

    Google Scholar 

  2. Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP, Strasser AWM (1992) Heterologous gene expression in Hoansneula polymrpha: efficient secretion of glucoamylase. Bio/Technology 9:291–295

    Article  Google Scholar 

  3. Schaefer S, Piontek M, Ahn S-J, Papendieck A, Janowicz ZA, Gellissen G (2001) Recombinant hepatitis B vaccines-characterization of the viral disease and vaccine production in the methylotrophic yeast Hansenula polymorpha. In: Dembowsky K, Stadler P (eds) Novel therapeutic proteins: selected case studies. Wiley, Weinheim, pp 245–276

    Google Scholar 

  4. Ryabova OB, Chmil OM, Sibirny AA (2003) Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha. FEMS Yeast Res 4:157–164

    Article  PubMed  CAS  Google Scholar 

  5. Ishchuk OP, Voronovsky AY, Abbas CA, Sibirny AA (2009) Construction of Hansenula polymorpha strains with improved thermotolerance. Biotechnol Bioeng 104:911–919

    Article  PubMed  CAS  Google Scholar 

  6. Grabek-Lejko D, Kurylenko OO, Sibirny VA, Ubiyvovk VM, Penninckx M, Sibirny AA (2011) Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione. J Ind Microbiol Biotechnol 38:1853–1859

    Article  PubMed  CAS  Google Scholar 

  7. Vigh L, Maresca B, Harwood JL (1998) Does the membrane’s physical state control the expression of heat shock and other genes? Trends Biochem Sci 23:369–374

    Article  PubMed  CAS  Google Scholar 

  8. Vigh L, Escriba PV, Sonnleitner A, Sonnleitner M, Piotto S, Maresca B, Horva′th I, Harwood JL (2005) The significance of lipid composition for membrane activity: new concepts and ways of assessing function. Prog Lipid Res 44:303–344

    Article  PubMed  CAS  Google Scholar 

  9. Cheawchanlertfa P, Cheevadhanarak S, Tanticharoen M, Maresca B, Laoteng K (2011) Up-regulated expression of desaturase genes of Mucor rouxii in response to low temperature associates with pre-existing cellular fatty acid constituents. Mol Biol Rep 38:3455–3462

    Article  PubMed  CAS  Google Scholar 

  10. Kajiwara S, Shirai A, Fujii T, Toguri T, Nakamura K, Ohtaguchi K (1996) Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana. Appl Environ Microbiol 62:4309–4313

    PubMed  CAS  Google Scholar 

  11. Rodriguez-Vargas S, Sanchez-Garcia A, Martinez-Rivas JM, Prieto JA, Randez-Gil F (2007) Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress. Appl Environ Microbiol 73:110–116

    Article  PubMed  CAS  Google Scholar 

  12. Cipak A, Jaganjac M, Tehlivets O, Kohlwein SD, Zarkovic N (2008) Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast. Biochim Biophys Acta 1781:283–287

    Article  PubMed  CAS  Google Scholar 

  13. Yazawa H, Iwahashi H, Kimura K, Kamisaka Y, Uemura H (2009) Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance. Yeast 26:167–184

    Article  PubMed  CAS  Google Scholar 

  14. Cipak A, Hasslacher M, Tehlivets O, Collinson EJ, Zivkovic M, Matijevic T, Wonisch W, Waeg G, Dawes IW, Zarkovic N, Kohlwein SD (2006) Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation. Free Radic Biol Med 40:897–906

    Article  PubMed  CAS  Google Scholar 

  15. Ruenwai R, Neiss A, Laoteng K, Vongsangnak W, Dalfard AB, Cheevadhanarak S, Petranovic D, Nielsen J (2011) Heterologous production of polyunsaturated fatty acids in Saccharomyces cerevisiae causes a global transcriptional response resulting in reduced proteasomal activity and increased oxidative stress. Biotechnol J 6:343–356

    Article  PubMed  CAS  Google Scholar 

  16. Anamnart S, Tolstorukov I, Kaneko Y, Harashima S (1998) Fatty acid desaturation in methylotrophic yeast Hansenula polymorpha strain CBS1976 and unsaturated fatty acid auxotrophic mutants. J Ferment Bioeng 85:476–482

    Article  CAS  Google Scholar 

  17. Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4:293–303

    Article  CAS  Google Scholar 

  18. Kyte J, Doolittle RR (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Article  PubMed  CAS  Google Scholar 

  19. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  20. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed  CAS  Google Scholar 

  21. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  22. Haan GJ, van Dijk R, Kiel JAKW, Veenhuis M (2001) Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration. FEMS Yeast Res 2:17–24

    Google Scholar 

  23. Stearns T, Ma H, Botstein D (1990) Manipulating yeast genome using plasmid vectors. Methods Enzymol 185:280–297

    Article  CAS  Google Scholar 

  24. Lepage G, Roy CC (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res 25:1391–1396

    PubMed  CAS  Google Scholar 

  25. Sakamoto T, Wada H, Nishida I, Ohmori M, Murata N (1994) Identification of conserved domains in the delta 12 desaturases of cyanobacteria. Plant Mol Biol 24:643–650

    Article  PubMed  CAS  Google Scholar 

  26. Sakuradani E, Kobayashi M, Ashikari T, Shimizu S (1999) Identification of delta 12-fatty acid desaturase from arachidonic acid-producing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Eur J Biochem 261:812–820

    Article  PubMed  CAS  Google Scholar 

  27. Wei D, Li M, Zhang X, Ren Y, Xing L (2004) Identification and characterization of a novel delta 12-fatty acid desaturase gene from Rhizopus arrhizus. FEBS Lett 573:45–50

    Article  PubMed  CAS  Google Scholar 

  28. Wei DSh, Li MCh, Zhang XX, Zhou H, Xing LJ (2006) A novel delta 12-fatty acid desaturase gene from methylotrophic yeast Pichia pastoris GS115. Acta Biochim Pol 53:753–759

    PubMed  CAS  Google Scholar 

  29. Wright MH, Heal WP, Mann DJ, Tate EW (2010) Protein myristoylation in health and disease. J Chem Biol 3:19–35

    Article  PubMed  Google Scholar 

  30. Duronio RJ, Rudnick DA, Johnson RL, Linder ME, Gordon JI (1991) Myristic acid auxotrophy caused by mutation of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. J Cell Biol 113:1313–1330

    Article  PubMed  CAS  Google Scholar 

  31. Duronio RJ, Knoll LJ, Gordon JI (1992) Isolation of a Saccharomyces cerevisiae long chain fatty acyl: CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation. J Cell Biol 117:515–529

    Article  PubMed  CAS  Google Scholar 

  32. Johnson DR, Knoll LJ, Levin DE, Gordon JI (1994) Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism. J Cell Biol 127:751–762

    Article  PubMed  CAS  Google Scholar 

  33. Nakagawa Y, Sakumoto N, Kaneko Y, Harashima S (2002) Mga2p is a putative sensor for low temperature and oxygen to induce OLE1 transcription in Saccharomyces cerevisiae. Biochem Biophys Res Commun 291:707–713

    Article  PubMed  CAS  Google Scholar 

  34. Alexandre H, Rousseaux I, Charpentier C (1994) Ethanol adaptation mechanisms in Saccharomycescerevisiae. Biotechnol Appl Biochem 20:173–183

    PubMed  CAS  Google Scholar 

  35. Piper PW (1995) The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap. FEMS Microbiol Lett 134:121–127

    Article  PubMed  CAS  Google Scholar 

  36. Peyou-Ndi MM, Watts JL, Browse J (2000) Identification and characterization of an animal Δ12 fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae. Arch Biochem Biophys 376:399–408

    Article  PubMed  CAS  Google Scholar 

  37. Xiao D, Wu S, Zhu X, Chen Y, Guo X (2010) Effects of soya fatty acids on cassava ethanol fermentation. Appl Biochem Biotechnol 160:410–420

    Article  PubMed  CAS  Google Scholar 

  38. Laoteng K, Jitsue S, Dandusitapunth Y, Cheevadhanarak S (2008) Ethanol-induced changes in expression profiles of cell growth, fatty acid and desaturase genes of Mucor rouxii. Fungal Genet Biol 45:61–67

    Article  PubMed  CAS  Google Scholar 

  39. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316

    Article  PubMed  CAS  Google Scholar 

  40. Costa V, Moradas-Ferreira P (2001) Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases. Mol Aspects Med 22:217–246

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by a Grant from Office of National Research Council of Thailand. We are grateful to Prof. Amorn Petsom for his critical comment on this study. We also thank Tayvich Vorapreeda for helping in figure preparation.

Author information

Authors and Affiliations

  1. The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, 10330, Thailand

    Sarintip Sooksai

  2. National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathum Thani, 12120, Thailand

    Pattsarun Chewchanlertfa & Kobkul Laoteng

  3. Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, 565-0871, Japan

    Yoshinobu Kaneko & Satoshi Harashima

Authors
  1. Sarintip Sooksai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pattsarun Chewchanlertfa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshinobu Kaneko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satoshi Harashima
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kobkul Laoteng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kobkul Laoteng.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 38 kb)

Supplementary material 2 (DOC 33 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sooksai, S., Chewchanlertfa, P., Kaneko, Y. et al. Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis. Mol Biol Rep 40, 4935–4945 (2013). https://doi.org/10.1007/s11033-013-2594-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-013-2594-3

Keywords

Search

Navigation