Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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


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.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 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

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

  3. 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

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

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

  6. 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

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

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

  9. 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

  10. 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

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

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

  13. 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

  14. 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

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

  16. 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

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

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

  19. 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

  20. 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

  21. 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

  22. 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

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

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

  25. 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

  26. 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

  27. 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

  28. 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

  29. 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

  30. 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

  31. 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

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

  33. 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

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

  35. 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

  36. 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

  37. 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

Download references


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.

Author information

Correspondence to Yuanxing Zhang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation


  • Reactive Oxygen Species
  • Fermentation
  • Intracellular Reactive Oxygen Species
  • Intracellular Reactive Oxygen Species Level
  • High Reactive Oxygen Species