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Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris


The yeast Pichia pastoris is a suitable production system for recombinant proteins due to its strong methanol-inducible AOX1 promoter. A key parameter of the production process is the specific methanol uptake rate. To control the methanol uptake and simultaneously maintain a constant methanol concentration during the production phase, two strategies were developed to generate purposeful oxygen limitation and to feed-forward control the specific methanol uptake rate into the optimum range. First, the cell density at induction was adjusted by prolonged preinduction glycerol feeding. Alternatively, the airflow rate was restricted and increased in parallel with the biomass. While the product accumulation started 20 h earlier with the first approach, the specific production rate of a single-chain antibody fragment was three times higher in the latter case. After 70 h of production, both schemes yielded product concentrations in the gram-per-liter range. Moreover, they release the requirement for dosage of pure oxygen and thereby can facilitate the scale-up of the production process. The different production profiles indicate that the impact of specific methanol uptake rate on protein production by recombinant P. pastoris depends on the control mode.

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  1. Bushell ME, Rowe M, Avignone-Rossa CA, Wardell JN (2003) Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris. Biotechnol Bioeng 82:678–683

  2. Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S-O (2005) Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 27:399–406

  3. Chiruvolu V, Cregg JM, Meagher MM (1997) Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations. Enzyme Microb Technol 21:277–283

  4. 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

  5. Cregg JM (1999) Expression in the methylotrophic yeast Pichia pastoris. In: Fernandez JM, Hoeffler JP (eds) Gene expression systems: using nature for the art of expression. Academic, San Diego, pp 157–191

  6. Cunha AE, Clemente JJ, Gomes R, Pinto F, Thomaz M, Miranda S, Pinto R, Moosmayer D, Donner P, Carrondo MJT (2004) Methanol induction optimization for scFv antibody fragment production in Pichia pastoris. Biotechnol Bioeng 86:458–467

  7. Curvers S, Brixius P, Klauser T, Thömmes J, Weuster-Botz D, Takors R, Wandrey C (2001) Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation. Biotechnol Prog 17:495–502

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

  9. Damasceno LM, Pla I, Chang H-J, Cohen L, Ritter G, Old LJ, Batt CA (2004) An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif 37:18–26

  10. Fischer R, Drossard J, Emans N, Commandeur U, Hellwig S (1999) Towards molecular farming in the future: Pichia pastoris-based production of single-chain antibody fragments. Biotechnol Appl Biochem 30:117–120

  11. Freyre FM, Vázquez JE, Ayala M, Canaán-Haden L, Bell H, Rodríguez I, González A, Cintado A, Gavilondo JV (2000) Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris. J Biotechnol 76:157–163

  12. Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H, Wan NC (2001) High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system. Biotechnol Bioeng 74:492–497

  13. Görgens JF, Passoth V, van Zyl WH, Knoetze JH, Hahn-Hägerdal B (2005) Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by Pichia stipitis. FEMS Yeast Res 5:677–683

  14. Hellwig S, Emde F, Raven NPG, Henke M, van der Logt P, Fischer R (2001) Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. Biotechnol Bioeng 74:344–352

  15. Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2003) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367–375

  16. Jahic M, Wallberg F, Bollok M, Garcia P, Enfors S-O (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial Cell Factories 2:6

  17. Khatri NK, Hoffmann F (2006) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng (in press)

  18. Klinner U, Fluthgraf S, Freese S, Passoth V (2004) Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis. Appl Microbiol Biotechnol 67:247–253

  19. Lee CY, Lee SJ, Jung KH, Katoh S, Lee EK (2003) High dissolved oxygen tension enhances heterologous protein expression by recombinant Pichia pastoris. Proc Biochem 38:1147–1154

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

  21. Lin Cereghino GP, Lin Cereghino J, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichiapastoris. Curr Opin Biotechnol 13:329–332

  22. Resina D, Cos O, Ferrer P, Valero F (2005) Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter. Biotechnol Bioeng 91:760–767

  23. Sinha J, Plantz BA, Zhang W, Gouthro M, Schlegel V, Liu C-P, Meagher MM (2003) Improved production of recombinant ovine Interferon-τ by Mut+ strain of Pichia pastoris using an optimized methanol feed profile. Biotechnol Prog 19:794–802

  24. Stratton J, Chiruvolu V, Meagher M (1998) High cell-density fermentation. In: Higgins DR, Cregg JM (eds) Pichia protocols. Humana, Totowa, pp 107–120

  25. Trentmann O, Khatri NK, Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20:1766–1775

  26. Trinh LB, Phue JN, Shiloach J (2003) Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris. Biotechnol Bioeng 82:438–444

  27. Vijayasankaran N, Carlson R, Srienc F (2005) Synthesis of poly[(R)-3-hydroxybutyric acid) in the cytoplasm of Pichia pastoris under oxygen limitation. Biomacromolecules 6:604–611

  28. Watermah HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44

  29. Yurimoto H, Sakai Y, Kato N (2002) Methanol metabolism. In: Gellisen G (eds) Hansenula polymorpha. Biology and applications. Wiley-VCH, Weinheim, pp 61–75

  30. Zhang W, Bevins MA, Plantz BA, Smith LA, Meagher MM (2000) Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of Botulinum neurotoxin, Serotype A. Biotechnol Bioeng 70:1–8

  31. Zhang W, Smith LA, Plantz BA, Schlegel VL, Meagher MM (2002) Design of methanol feed control in Pichia pastoris fermentations based upon a growth model. Biotechnol Prog 18:1392–1399

  32. Zhou X-S, Zhang Y-X (2002) Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate. Biotechnol Lett 24:1449–1453

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Correspondence to Frank Hoffmann.

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Khatri, N.K., Hoffmann, F. Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris . Appl Microbiol Biotechnol 72, 492–498 (2006). https://doi.org/10.1007/s00253-005-0306-1

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  • Recombinant Protein Production
  • Specific Production Rate
  • Specific Uptake Rate
  • Final Product Concentration
  • High Methanol Concentration