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Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation

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Abstract

In recombinant Pichia pastoris fermentation for hirudin production, copious cells were not viable and most of the secreted hirudin molecules were C-terminally truncated at the end of fermentation. In this work, the influences of reactive oxygen species (ROS) on cell viability and hirudin production were subsequently studied. In contrast to the untreated control condition, the addition of ascorbic acid at the methanol fed-batch phase could obviously relieve the damage of intracellular ROS to cell membranes. As a result, the cell viability could be increased to 91% from 74% in control at the end of fermentation and the extracellular proteolysis of hirudin reduced. Intact and total hirudin production, by supplying ascorbic acid, could reach 2.90 and 5.03 g/l, respectively, in contrast to 1.75 and 4.70 g/l at the control condition. Ascorbic acid, 4 mmol/l or more, in the fermentation broth increased markedly the production of the intact hirudin, despite a little effect on total hirudin production.

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References

  • Avaro S, Belgaze-Touze N, Sibella-Arguelles C, Haguenauer-Tsapis R (2002) Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants. Yeast 19:351–371

    Article  CAS  Google Scholar 

  • Belo I, Pinheiro R, Mota M (2005) Morphological and physiological changes in Saccharomycescerevisiae by oxidative stress from hyperbaric air. J Biotechnol 115:397–404

    Article  CAS  Google Scholar 

  • Brierley RA, Bussineau C, Kossan R, Melton A, Siegel RS (1990) Fermentation development of recombinant Pichia pastoris expressing the heterologous gene: bovine lysozyme. Ann N Y Acad Sci 589:350–362

    Article  CAS  Google Scholar 

  • Burgeois C (1992) Determination of vitamin E: tocopherols and tocotrienols. Elsevier Applied Science, New York

    Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  Google Scholar 

  • Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholz RG, Madden KR, Kellaris PA, Davis GR, Smiley BL, Cruze J, Torregrossa R, Velicelebi G, Thill GP (1987) High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Bio/Technol 5:479–485

    CAS  Google Scholar 

  • Cregg JM, Vedvick TS, Raschke WC (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technol 11:905–910

    CAS  Google Scholar 

  • Davey HM (2002) Flow cytometric techniques for the detection of microorganisms. Methods Cell Sci 24:91–97

    Article  CAS  Google Scholar 

  • Digan ME, Lair SV, Brierley RA, Siegel RS, Williams ME, Ellis SB, Kellaris PA, Provow SA, Craig WS, Velicelebi G, Harplod MM, Thill GP (1989) Continuous production of a novel lysozyme via secretion from the yeast Pichia pastoris. Bio/Technol 7:160–164

    CAS  Google Scholar 

  • Douma AC, Veenhuis M, de Koning W, Evers M, Harder W (1985) Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol grown Hansenula polymorpha. Arch Microbiol 143:237–243

    Article  CAS  Google Scholar 

  • Gimenez JA, Monkovic DD, Dekleva ML (2000) Identification and monitoring of protease activity in recombinant Saccharomycescerevisiae. Biotechnol Bioeng 67:245–251

    Article  CAS  Google Scholar 

  • Grant D, Levine A, Dor-Hefetz E (2003) Sugar-induced apoptosis in yeast cells. FEMS Yeast Res 4:7–13

    Article  Google Scholar 

  • Hansen RJ, Switzer RL, Hinze H, Holzer H (1977) Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast. Biochim Biophys Acta 496:103–114

    Article  CAS  Google Scholar 

  • Heim J, Takabayashi K, Meyhack B, Marki W, Pohlig G (1994) C-Terminal proteolytic degradation of recombinant desulfatohirudin and its mutants in the yeast Saccharomyces cerevisiae. Eur J Biochem 226:341–353

    Article  CAS  Google Scholar 

  • Hilt W, Wolf DH (1992) Stress-induced proteolysis in yeast. Mol Microbiol 6:2437–2442

    Article  CAS  Google Scholar 

  • Hohenblum H, Borth N, Mattanovich D (2003) Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J Biotechnol 102:281–290

    Article  CAS  Google Scholar 

  • Hong F, Meinander NQ, Jönsson LJ (2002) Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnol Bioeng 79:438–449

    Article  CAS  Google Scholar 

  • Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomycescerevisiae. Yeast 14:1511–1527

    Article  CAS  Google Scholar 

  • Kim CH, Sohn JH, Choi ES, Rhee SK (1996) Effect of soybean oil on the enhanced expression of hirudin gene in Hansenulapolymorpha. Biotechnol Lett 18:417–422

    Article  CAS  Google Scholar 

  • Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T (2000) High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichiapastoris with minimal protease production and activation. J Biosci Bioeng 89:55–61

    Article  CAS  Google Scholar 

  • Lehman E, Joyce G, Bailey F, Markus Z, Schultz D, Dunwiddie T, Jacobson A, Miller J (1993) Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1–HV2 hirudin variant expressed in Saccharomycescerevisiae. Protein Expr Purif 4:247–255

    Article  CAS  Google Scholar 

  • Moraitis C, Curran BPG (2004) Reactive oxygen species may influence the heat shock response and stress tolerance in the yeast Saccharomyces cerevisiae. Yeast 21:313–323

    Article  CAS  Google Scholar 

  • Niki E (1987) Antioxidants in relation to lipid peroxidation. Chem Phys Lipids 44:227–253

    Article  CAS  Google Scholar 

  • Packer JE, Slater TF, Wilson RL (1979) Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737–738

    Article  CAS  Google Scholar 

  • Riehl-Bellon N, Dorothee C, Michele A, Alain VD, Magda M, Gerard L, Yves L, Stephen WB, Michael C, Carolyn R (1989) Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae. Biochemistry 28:2941–2949

    Article  CAS  Google Scholar 

  • Rosenfeld SA, Nadeau D, Tirado J, Hollis GF, Knabb RM, Jia S (1996) Production and purification of recombinant hirudin expression in the methylotrophic yeast Pichia pastoris. Protein Exp Purif 8:476–482

    Article  CAS  Google Scholar 

  • Sinha J, Plantz BA, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-τ. Biotechnol Bioeng 89:102–112

    Article  CAS  Google Scholar 

  • Sohn JH, Choi ES, Chung BH, Youn DJ, Seo JH, Rhee SK (1995) Process development for the production of recombinant hirudin in Saccharomyces cerevisiae: from upstream to downstream. Process Biochem 30:653–660

    Article  CAS  Google Scholar 

  • Storz G, Christman MF, Sies H, Ames BN (1987) Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium. Proc Natl Acad Sci U S A 84:8917–8921

    Article  CAS  Google Scholar 

  • Walrand S, Valeix S, Rodriguez C, Ligot P, Chassagne J, Vasson MP (2003) Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes. Clin Chim Acta 331:103–110

    Article  CAS  Google Scholar 

  • Weydemann U, Keup P, Piontek M, Strasser AW, Schweden J, Gellissen G, Janowicz ZA (1995) High-level secretion of hirudin by Hansenula polymorpha authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44:377–385

    Article  CAS  Google Scholar 

  • Wolff SP, Garner A, Dean RT (1986) Free radicals, lipids and protein degradation. Trends Biochem Sci 11:27–31

    Article  CAS  Google Scholar 

  • Yang JZ, Zhou XS, Zhang YX (2004) Improvement of recombinant hirudin production by controlling NH4 + concentration in Pichia pastoris fermentation. Biotechnol Lett 26:1013–1017

    Article  CAS  Google Scholar 

  • Yasuhara T, Nakai T, Ohashi A (1994) Aminopeptidase Y, a new aminopeptidase from Saccharomyces cerevisiae. J Biol Chem 269:13644–13650

    CAS  PubMed  Google Scholar 

  • Zhou XS, Zhang YX (2002) Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate. Biotechnol Lett 24:1449–1453

    Article  CAS  Google Scholar 

  • Zhou WB, Zhang YX (2004) Purification and identification of recombinant hirudin and its degradation derivatives expressed in Pichia pastoris. Prep Biochem Biotechnol 34:239–252

    Article  CAS  Google Scholar 

  • Zhou XS, Lu J, Fan WM, Zhang YX (2002) Development of a responsive methanol sensor and its application in Pichia pastoris fermentation. Biotechnol Lett 24:643–646

    Article  CAS  Google Scholar 

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Acknowledgements

We are most grateful to Dr. Ping Shi, who has helped us so much during the FCM measure. Our thanks also go to Mr. Jizhong Yang for his valuable suggestions and advice on the experimental work.

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  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Anfeng Xiao, Xiangshan Zhou, Li Zhou & Yuanxing Zhang

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  1. Anfeng Xiao
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  2. Xiangshan Zhou
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  3. Li Zhou
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  4. Yuanxing Zhang
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Correspondence to Yuanxing Zhang.

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Xiao, A., Zhou, X., Zhou, L. et al. Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol 72, 837–844 (2006). https://doi.org/10.1007/s00253-006-0338-1

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  • DOI: https://doi.org/10.1007/s00253-006-0338-1

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