Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

  • 629 Accesses

  • 34 Citations


A growth-associated model was applied to the production of recombinant ovine interferon-τ (rOvIFN-τ) with Pichia pastoris for the purpose of manufacturing preclinical and clinical active material. This model predicts that product yields will be the greatest when the specific growth of the culture is maintained at a steady and optimal rate. However, rOvIFN-τ yields did not meet the expected linear model but most closely corresponded to a polynomial relationship. After transitioning from glycerol to methanol, product accumulated for 31–45 h, and then the yield decreased. This production shift, which has been termed decoupling, was clearly related to time on methanol and not culture density. It was determined that a correlation exists between the decoupling point and a drop in energy state of the cell when expressing β-galactosidase. By assigning decoupling as a constraint that limits productivity and by reformulating the growth medium, the time prior to decoupling increased to 46.8±2.4 h, product yield improved for rOvIFN-τ from 203 to 337 mg l−1, and the coefficient of variation for yield decreased from 67.9 to 23.3%. A robust and stable fermentation process was realized, resulting in a 210% improvement in total yield from 557±357 to 1,172±388 mg.

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

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


  1. Atkinson D, Walton G (1967) Adenosine triphosphate conservation in metabolic regulation: rate liver citrate cleavage enzyme. J Biol Chem 242:3239–3241

  2. Babul J, Clifton D, Kretschmer M, Fraenkel D (1993) Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase. Biochemistry 32:4685–4692

  3. Brady C, Shimp R, Miles A, Whitmore M, Stowers A (2001) High-level production and purification of P20P2MSP119, an important vaccine antigen for malaria, expressed in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 23:468–475

  4. Byrne M, Titball R, Holley J, Smith L (2000) Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris. Protein Expr Purif 18:327–337

  5. Chuang S-M, Chen L, Lambertson D, Anand M, Kinzy TG, Madura K (2005) Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A. Mol Cell Biol 25:403–413

  6. Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385

  7. Cregg JM, Tschopp J, Stillman C, Siegel R, Akong M, Craig W, Buckholz R, Madden K, Kellaris P, Davis G, Smiley B, Cruze J, Torregrossa R, Velice-lebi G, Thill G (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/technology 5:479–485

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

  9. Edwards K, Urban J, Schreiber G (1979) Relationship between protein synthesis and secretion in liver cells and the state of the adenine nucleotide system. Aust J Biol Sci 32:299–307

  10. Files D, Ogawa M, Scaman C, Baldwin S (2001) A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C. Enzyme Microb Technol 29:335–340

  11. Gleeson M, White C, Meininger D, Komives E (1998) Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol Biol 103:81–94

  12. Jamieson J, Palade G (1968) Intracellular transport of secretory proteins in the pancreatic exocrine cell IV: metabolic requirements. J Cell Biol 39:589–603

  13. Katakura Y, Zhang W, Guoqiang O, Takeshi K, Kishimoto M, Goto Y, Suga K-I (1998) Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. J Ferment Bioeng 86:482–487

  14. Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Tomomitsu K (2000) High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategy. J Biosci Bioeng 90:280–288

  15. Li Z, Xiong F, Lin Q, d’Anjou M, Daugulis AJ, Yang DS, Hew CL (2001) Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif 21:438–445

  16. Lin-Cereghino J, Geoff P, Ilgen C, Cregg JM (2002) Production of recombinant proteins in ferment or cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332

  17. Miller J (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory, Woodbury, pp 72–74

  18. Plantz B, Andersen J, Smith L, Meagher M, Schlegel V (2003) Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths. J Ind Microbiol Biotech 30:643–650

  19. Sinha J, Plantz B, Zhang W, Gouthro M, Schlegel V, Liu C-P, Meagher M (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

  20. Sinha J, Plantz B, Inan M, Meagher M (2004) Causes of proteolytic degradation of secreted recombinant proteins production in methylotrophic yeast Pichia pastoris—case study with recombinant ovine interferon-τ. Biotechnol Bioeng 89:102–112

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

  22. 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

  23. Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15:3859–3867

  24. US Food and Drug Administration (2005) Code of federal regulations. General biological product standards, title 21, parts 610.9 and 610.13. US Government Printing Office, Washington, DC

  25. Van Heeke G, Ott TL, Strauss A, Ammaturo D, Bazer FW (1996) High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris. J Interferon Cytokine Res 16:119–126

  26. Velkov VV, Matys VY, Sokolov DM (1999) How overproduction of foreign proteins affects physiology of the recombinant strains of Hansenula polymorpha. J Biosci 24:279–286

  27. Wood T, Peretti S (1990) Depression of protein synthetic capacity due to cloned-gene expression in E. coli. Biotechnol Bioeng 36:865–877

  28. Zhang W, Inan M, Meagher M (2000a) Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris. Biotechnol Bioprocess Eng 5:275–287

  29. Zhang W, Bevins M, Plantz B, Smith L, Meagher M (2000b) 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

  30. Zhang W, Smith L, Plantz B, Schlegel V, Meagher (2002) Design of methanol feed control in Pichia pastoris fermentations based upon a growth model. Biotechnol Prog 18:1392–1399

  31. Zhang W, Hywood Potter KJ, Plantz BA, Schlegel VL, Smith LA, Meagher MM (2003) Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. J Ind Microbiol Biotechnol 30:210–215

Download references


The funding for this research was generously supported by a grant provided by the Pepgen Corporation. We further acknowledge Dr. Gautam Sarath for data analysis, and the staff of the University of Nebraska Biological Process Development Facility.

Author information

Correspondence to Vicki L. Schlegel.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Plantz, B.A., Sinha, J., Villarete, L. et al. Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl Microbiol Biotechnol 72, 297–305 (2006).

Download citation


  • Fermentation
  • Culture Density
  • Adenylate Energy Charge
  • AOX1 Promoter
  • Pastoris Expression System