Efficient Development of a Mixed Feed Process for Pichia pastoris

  • David Johannes Wurm
  • Oliver SpadiutEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)


Pichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. A prominent promoter system for recombinant protein production in P. pastoris is the promoter of alcohol oxidase (PAOX1) which is induced by methanol, but repressed by several other carbon sources, like glucose and glycerol. Thus, typical cultivation strategies for such P. pastoris strains describe two different phases: growth on a carbon source, like glycerol, to get a high biomass concentration, followed by the induction of recombinant protein production by methanol. However, cells barely grow on methanol resulting in only moderate productivity in such bioprocesses. To enhance productivity, it is common to employ mixed substrate feeding strategies. The knowledge of certain strain-specific parameters is required to be able to set up such mixed feed fed-batch cultivations to avoid methanol accumulation and guarantee highest productivity. Here, we present an efficient strategy comprising only one experiment to determine the settings of such a mixed feed system based on the physiology of the respective yeast strain.

Key words

Pichia pastoris Methanol pulse Specific substrate uptake rate Dynamic fed-batch strategy Strain characterization 


  1. 1.
    Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270CrossRefGoogle Scholar
  2. 2.
    Weinacker D, Rabert C, Zepeda AB, Figueroa CA, Pessoa A, Farias JG (2013) Applications of recombinant Pichia pastoris in the healthcare industry. Braz J Microbiol 44:1043–1048CrossRefGoogle Scholar
  3. 3.
    Spohner SC, Muller H, Quitmann H, Czermak P (2015) Expression of enzymes for the usage in food and feed industry with Pichia pastoris. J Biotechnol 202:118–134CrossRefGoogle Scholar
  4. 4.
    Cregg JM (2007) Pichia protocols, Methods Mol Biol, 2nd edn. Springer, New York, p 268Google Scholar
  5. 5.
    Cregg JM, Cereghino JL, Shi JY, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52CrossRefGoogle Scholar
  6. 6.
    Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66CrossRefGoogle Scholar
  7. 7.
    Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332CrossRefGoogle Scholar
  8. 8.
    Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138CrossRefGoogle Scholar
  9. 9.
    Daly R, Hearn MT (2006) Expression of the human activin type I and II receptor extracellular domains in Pichia pastoris. Protein Expr Purif 46:456–467CrossRefGoogle Scholar
  10. 10.
    Spadiut O, Herwig C (2014) Dynamics in bioprocess development for Pichia pastoris. Bioengineering 5:401–404Google Scholar
  11. 11.
    Zhang WH, Potter KJH, 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 30:210–215CrossRefGoogle Scholar
  12. 12.
    Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY, Tu FZ (2009) Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep 36:1611–1619CrossRefGoogle Scholar
  13. 13.
    Zalai D, Dietzsch C, Herwig C, Spadiut O (2012) A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding. Biotechnol Prog 28:878–886CrossRefGoogle Scholar
  14. 14.
    Arnau C, Casas C, Valero F (2011) The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system. Biochem Eng J 57:30–37CrossRefGoogle Scholar
  15. 15.
    d’Anjou MC, Daugulis AJ (2001) A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol Bioeng 72:1–11CrossRefGoogle Scholar
  16. 16.
    Jungo C, Marison I, von Stockar U (2007) Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: a quantitative study based on concentration gradients in transient continuous cultures. J Biotechnol 128:824–837CrossRefGoogle Scholar
  17. 17.
    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 74:344–352CrossRefGoogle Scholar
  18. 18.
    Capone S, Horvat J, Herwig C, Spadiut O (2015) Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter. Microb Cell Factories 14:101CrossRefGoogle Scholar
  19. 19.
    Spadiut O, Zalai D, Dietzsch C, Herwig C (2013) Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris. Bioprocess Biosyst Eng 37:1163–1172CrossRefGoogle Scholar
  20. 20.
    Finn B, Harvey LM, McNeil B (2006) Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker’s yeast fed-batch bioprocess. Yeast 23:507–517CrossRefGoogle Scholar
  21. 21.
    Fehrenbach R, Comberbach M, Petre JO (1992) On-line biomass monitoring by capacitance measurement. J Biotechnol 23:303–314CrossRefGoogle Scholar
  22. 22.
    Sagmeister P, Langemann T, Wechselberger P, Meitz A, Herwig C (2013) A dynamic method for the investigation of induced state metabolic capacities as a function of temperature. Microb Cell Factories 12:94CrossRefGoogle Scholar
  23. 23.
    Wechselberger P, Sagmeister P, Herwig C (2013) Real-time estimation of biomass and specific growth rate in physiologically variable recombinant fed-batch processes. Bioprocess Biosyst Eng 36:1205–1218CrossRefGoogle Scholar
  24. 24.
    Dietzsch C, Spadiut O, Herwig C (2011) A fast approach to determine a fed batch feeding profile for recombinant Pichia pastoris strains. Microb Cell Factories 10:85CrossRefGoogle Scholar
  25. 25.
    Dietzsch C, Spadiut O, Herwig C (2011) A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris. Microb Cell Factories 10:14CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Chemical, Environmental and Bioscience Engineering, Research Division Biochemical Engineering, Vienna University of Technology (TU Wien)ViennaAustria

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