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

  • Applied Genetics and Molecular Biotechnology
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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.

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References

  • 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 116:321–335

    Article  Google Scholar 

  • 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 8:229–235

    Article  Google Scholar 

  • Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  CAS  Google Scholar 

  • d'Anjou MC, Daugulis AJ (2001) A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol Bioeng 72:1–11

    Article  Google Scholar 

  • 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 8:1380–1392

    Article  CAS  Google Scholar 

  • 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 8:179

    Article  Google Scholar 

  • 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 7:11

    Article  Google Scholar 

  • Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750

    Article  CAS  Google Scholar 

  • Haynes CM, Titus EA, Cooper AA (2004) Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death. Mol Cell 15:767–776

    Article  CAS  Google Scholar 

  • 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 85:367–375

    Article  CAS  Google Scholar 

  • 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 54:234–239

    Article  CAS  Google Scholar 

  • Inan M, Aryasomayajula D, Sinha J, Meagher MM (2006) Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnol Bioeng 93:771–778

    Article  CAS  Google Scholar 

  • 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 39:936–944

    Article  CAS  Google Scholar 

  • 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 130:236–246

    Article  CAS  Google Scholar 

  • 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 11:59–69

    Article  CAS  Google Scholar 

  • Kohrer K, Domdey H (1991) Preparation of high molecular weight RNA. Methods Enzymol 194:398–405

    Google Scholar 

  • 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 326:817–824

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Ma Y, Hendershot LM (2001) The unfolding tale of the unfolded protein response. Cell 107:827–830

    Article  CAS  Google Scholar 

  • 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 105:18525–18530

    Article  CAS  Google Scholar 

  • 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 27:339–345

    Article  CAS  Google Scholar 

  • Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135

    Article  CAS  Google Scholar 

  • Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305

    CAS  Google Scholar 

  • Perrone GG, Tan SX, Dawes IW (2008) Reactive oxygen species and yeast apoptosis. Biochim Biophys Acta 1783:1354–1368

    Article  CAS  Google Scholar 

  • 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 6:21

    Article  Google Scholar 

  • 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 12:181–184

    Article  CAS  Google Scholar 

  • 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 330:39–47

    Article  CAS  Google Scholar 

  • 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 101:249–258

    Article  CAS  Google Scholar 

  • 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 116:251–260

    Article  CAS  Google Scholar 

  • 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 21:71–80

    Article  CAS  Google Scholar 

  • Wen SH, Zhang T, Tan TW (2005) Optimization of the amino acid composition in glutathione fermentation. Process Biochem 40:3474–3479

    Article  CAS  Google Scholar 

  • 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 72:837–844

    Article  CAS  Google Scholar 

  • 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 8:540–549

    Article  CAS  Google Scholar 

  • Yurimoto H, Kato N, Sakai Y (2005) Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism. Chem Rec 5:367–375

    Article  CAS  Google Scholar 

  • 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 31:679–684

    Article  CAS  Google Scholar 

  • 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 107:954–963

    Article  CAS  Google Scholar 

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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).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, P.O. Box 329, 130 Meilong Rd, Shanghai, 200237, People’s Republic of China

    Taicheng Zhu, Meijin Guo, Yingping Zhuang, Ju Chu & Siliang Zhang

  2. Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

    Taicheng Zhu

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  1. Taicheng Zhu
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  2. Meijin Guo
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  3. Yingping Zhuang
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Correspondence to Meijin Guo.

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Zhu, T., Guo, M., Zhuang, Y. et al. Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes. Appl Microbiol Biotechnol 89, 1127–1135 (2011). https://doi.org/10.1007/s00253-010-2944-1

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  • DOI: https://doi.org/10.1007/s00253-010-2944-1

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