Advertisement

Applied Microbiology and Biotechnology

, Volume 89, Issue 4, pp 1127–1135 | Cite as

Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes

  • Taicheng Zhu
  • Meijin GuoEmail author
  • Yingping Zhuang
  • Ju Chu
  • Siliang Zhang
Applied Genetics and Molecular Biotechnology

Abstract

Increased copy number of foreign gene can result in the alteration of normal metabolism in Pichia pastoris. To better understand the effect of foreign gene dosage on the cellular physiology of P. pastoris cells, comparative transcriptional analysis was performed among three P. pastoris strains carrying 0, 6, and 18 copies of porcine insulin precursor (PIP) expression cassettes, respectively. mRNA levels of 13 selected genes involved in methanol metabolic pathway, central metabolic pathway, protein folding, and oxidative stress were determined by real-time PCR. Results showed that enhanced PIP copy number resulted in an increase in PIP mRNA and also in folding stress on the yeast cells’ endoplasmic reticulum. The metabolism of 6-copy P. pastoris strain was not significantly changed as compared to 0-copy strain (control). In contrast, physiology of 18-copy strain was remarkably affected, characterized by the upregulation of antioxidative genes and readjusted expression level of methanol metabolic pathway genes. These data suggested that high copy P. pastoris strain might be suffering from protein folding-related oxidative stress and insufficient supply of carbon and energy sources.

Keywords

Gene dosage Pichia pastoris Methanol metabolism Cell physiology mRNA quantitative analysis 

Notes

Acknowledgments

We thank Professor James M. Gregg (Keck Graduate Institute of Applied Life Sciences) for providing sequence information. This research was supported by a grant from National Natural Science Foundation of China (no. 30560006) and by National Basic Research Program of China (no. 2007CB714303).

References

  1. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F (2005) Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol 116:321–335CrossRefGoogle Scholar
  2. Curvers S, Linnemann J, Klauser T, Wandrey C, Takors R (2002) Recombinant protein production with Pichia pastoris in continuous fermentation—kinetic analysis of growth and product formation. Eng Life Sci 8:229–235CrossRefGoogle Scholar
  3. Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138CrossRefGoogle Scholar
  4. d'Anjou MC, Daugulis AJ (2001) A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol Bioeng 72:1–11CrossRefGoogle Scholar
  5. Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392CrossRefGoogle Scholar
  6. Gasser B, Maurer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttila M, Mattanovich D (2007) Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics 8:179CrossRefGoogle Scholar
  7. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodriguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A (2008) Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 7:11CrossRefGoogle Scholar
  8. Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750CrossRefGoogle Scholar
  9. Haynes CM, Titus EA, Cooper AA (2004) Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death. Mol Cell 15:767–776CrossRefGoogle Scholar
  10. Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367–375CrossRefGoogle Scholar
  11. Huo XD, Liu YY, Wang X, Ouyang PK, Niu ZD, Shi YH, Qiu BS (2007) Co-expression of human protein disulfide isomerase (hPDI) enhances secretion of bovine follicle-stimulating hormone (bFSH) in Pichia pastoris. Protein Expr Purif 54:234–239CrossRefGoogle Scholar
  12. Inan M, Aryasomayajula D, Sinha J, Meagher MM (2006) Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnol Bioeng 93:771–778CrossRefGoogle Scholar
  13. Jungo C, Rerat C, Marison IW, Von Stockar U (2006) Quantitative characterization of the regulation of the synthesis of alcohol oxidase and of the expression of recombinant avidin in a Pichia pastoris Mut+ strain. Enzyme Microb Technol 39:936–944CrossRefGoogle Scholar
  14. Jungo C, Marison I, von Stockar SU (2007) Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures. J Biotechnol 130:236–246CrossRefGoogle Scholar
  15. Kimata Y, Ishiwata-Kimata Y, Yamada S, Kohno K (2006) Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes Cells 11:59–69CrossRefGoogle Scholar
  16. Kohrer K, Domdey H (1991) Preparation of high molecular weight RNA. Methods Enzymol 194:398–405Google Scholar
  17. Liu SH, Chou WI, Sheu CC, Chang MD (2005) Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations. Biochem Biophys Res Commun 326:817–824CrossRefGoogle Scholar
  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ method. Methods 25:402–408CrossRefGoogle Scholar
  19. Ma Y, Hendershot LM (2001) The unfolding tale of the unfolded protein response. Cell 107:827–830CrossRefGoogle Scholar
  20. Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, Kaufman RJ (2008) Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci 105:18525–18530CrossRefGoogle Scholar
  21. Mansur M, Cabello C, Hernandez L, Pais J, Varas L, Valdes J, Terrero Y, Hidalgo A, Plana L, Besada V, Garcia L, Lamazares E, Castellanos L, Martinez E (2005) Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol Lett 27:339–345CrossRefGoogle Scholar
  22. Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135CrossRefGoogle Scholar
  23. Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305Google Scholar
  24. Perrone GG, Tan SX, Dawes IW (2008) Reactive oxygen species and yeast apoptosis. Biochim Biophys Acta 1783:1354–1368CrossRefGoogle Scholar
  25. Resina D, Bollok M, Khatri NK, Valero F, Neubauer P, Ferrer P (2007) Transcriptional response of Pichia pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction. Microb Cell Fact 6:21CrossRefGoogle Scholar
  26. Scorer CA, Clare JJ, Mccombie WR, Romanos MA, Sreekrishna K (1994) Rapid selection using g418 of high copy number transformants of Pichia pastoris for high-level foreign gene-expression. Biotechnology 12:181–184CrossRefGoogle Scholar
  27. Sunga AJ, Cregg JM (2004) The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris. Gene 330:39–47CrossRefGoogle Scholar
  28. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258CrossRefGoogle Scholar
  29. Vad R, Nafstad E, Dahl LA, Gabrielsen OS (2005) Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol 116:251–260CrossRefGoogle Scholar
  30. Vassileva A, Chugh DA, Swaminathan S, Khanna N (2001) Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 21:71–80CrossRefGoogle Scholar
  31. Wen SH, Zhang T, Tan TW (2005) Optimization of the amino acid composition in glutathione fermentation. Process Biochem 40:3474–3479CrossRefGoogle Scholar
  32. Xiao AF, Zhou XS, Zhou L, Zhang YX (2006) Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol 72:837–844CrossRefGoogle Scholar
  33. Yano T, Takigami E, Yurimoto H, Sakai Y (2009) Yap1-regulated glutathione redox system curtails accumulation of formaldehyde and reactive oxygen species in methanol metabolism of Pichia pastoris. Eukaryot Cell 8:540–549CrossRefGoogle Scholar
  34. Yurimoto H, Kato N, Sakai Y (2005) Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism. Chem Rec 5:367–375CrossRefGoogle Scholar
  35. Zhu T, Guo M, Sun C, Qian J, Zhuang Y, Chu J, Zhang S (2009a) A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett 31:679–684CrossRefGoogle Scholar
  36. Zhu T, Guo M, Tang Z, Zhang M, Zhuang Y, Chu J, Zhang S (2009b) Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris. J Appl Microbiol 107:954–963CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Taicheng Zhu
    • 1
    • 2
  • Meijin Guo
    • 1
    Email author
  • Yingping Zhuang
    • 1
  • Ju Chu
    • 1
  • Siliang Zhang
    • 1
  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiPeople’s Republic of China
  2. 2.Institute of Microbiology, Chinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations