Skip to main content
SpringerLink
Log in

A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation

  • Fermentation, Cell Culture and Bioengineering - Short Communication
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (µ) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same µ at either 22 or 30 °C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10–10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

References

  1. 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 57:30–37

    Article  CAS  Google Scholar 

  2. Arnau C, Ramón R, Casas C, Valero F (2010) Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess. Enzyme Microb Technol 46:494–500

    Article  CAS  PubMed  Google Scholar 

  3. Berlec A, Strukelj B (2013) Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells. J Ind Microbiol Biotechnol 40:257–274

    Article  CAS  PubMed  Google Scholar 

  4. Berrios J, Altamirano C, Osses N, Gonzalez R (2011) Continuous CHO cell cultures with improved recombinant protein productivity by using mannose as carbon source: metabolic analysis and scale-up simulation. Chem Eng Sci 66:2431–2439

    Article  CAS  Google Scholar 

  5. Çalik P, Bozkurt B, Zerze GH, Inankur B, Bayraktar E, Boy E, Orman MA, Açik E, Özdamar TH (2013) Effect of co-substrate sorbitol different feeding strategies on human growth hormone production by recombinant Pichia pastoris. J Chem Technol Biotechol 88:1631–1640

    Article  Google Scholar 

  6. Canales C, Altamirano C, Berrios J (2015) Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture. Biotechnol Prog 31:707–714

    Article  CAS  PubMed  Google Scholar 

  7. Carly F, Niu H, Delvigne F, Fickers P (2016) Influence of methanol/sorbitol co-feeding rate on pAOX1 induction in a Pichia pastoris Mut + strain in bioreactor with limited oxygen transfer rate. J Ind Microbiol Biotechnol 43:517–523

    Article  CAS  PubMed  Google Scholar 

  8. Cos O, Ramón R, Montesinos JL, Valero F (2006) Operational strategies monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microb Cell Factor 5:17

    Article  Google Scholar 

  9. Cos O, Serrano A, Montecinos 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

    Article  PubMed  Google Scholar 

  10. Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392

    Article  CAS  PubMed  Google Scholar 

  11. Gao MJ, Lin Z, Yu RS et al (2012) Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift. Bioproc Biosyst Eng 35:1125–1136

    Article  CAS  Google Scholar 

  12. Gasser B, Mauer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttilä M, Mattanovich D (2007) Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genom 8:179

    Article  Google Scholar 

  13. He D, Luo W, Wang Z, Lv P, Yuan Z (2015) Combined use of GAP and AOX1 promoters and optimization of culture conditions to enhance expression of Rhizomucor miehei lipase. J Ind Microbiol Biotechnol 42:1175–1182

    Article  CAS  PubMed  Google Scholar 

  14. Huang CJ, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39:383–399

    Article  CAS  PubMed  Google Scholar 

  15. Imamoglu E, Sukan FV (2013) Scale-up and kinetic modeling for bioethanol production. Bioresour Technol 144:311–320

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  17. Jin HG, Liu X, Ye Z, Duan Z, Shi Z (2010) Enhanced porcine interferon-α production by recombinant Pichia pastoris with a combinational control strategy of low induction temperature and high dissolved oxygen concentration. Biochem Eng J 52:91–98

    Article  CAS  Google Scholar 

  18. 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 128:824–837

    Article  CAS  PubMed  Google Scholar 

  19. Jungo C, Schenk J, Pasquier M, Marison I, von Stockar U (2007) A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris. J Biotechnol 131:57–66

    Article  CAS  PubMed  Google Scholar 

  20. Junker BH (2004) Scale-up methodologies for Escherichia coli and yeast fermentation processes. J Biosci Bioeng 97:347–364

    Article  CAS  PubMed  Google Scholar 

  21. Liu WC, Zhu P (2015) Pilot studies on scale-up biocatalysis of 7-β-xylosyl-10-deacetyltaxol and its analogues by an engineered yeast. J Ind Microbiol Biotechnol 42:867–876

    Article  CAS  PubMed  Google Scholar 

  22. Montes F, Catalán J, Galán MA (1999) Prediction of kL a in yeast broths. Process Biochem 34:549–555

    Article  CAS  Google Scholar 

  23. Niu H, Daukandt M, Rodriguez C, Fickers P, Bogaerts P (2013) Dynamic modeling of methylotrophic Pichia pastoris culture with exhaust gas analysis: from cellular metabolism to process simulation. Chem Eng Sci 87:381–392

    Article  CAS  Google Scholar 

  24. Niu H, Jost L, Pirlot N, Sassi H, Daukandt M, Rodriguez C, Fickers P (2013) A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain. Microb Cell Fact 12:33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Potvin G, Ahmad A, Zhang Z (2010) Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: a review. Biochem Eng J 64:91–105

    Article  Google Scholar 

  26. Sagmeister P, Wechselberger P, Jazini M, Meitzb A, Langemann T, Herwig C (2013) Soft sensor assisted dynamic bioprocess control: efficient tools for bioprocess development. Chem Eng Sci 96:190–198

    Article  CAS  Google Scholar 

  27. Solà A, Jouhten P, Maaheimo H, Sánchez-Ferrando F, Szyperski T, Ferrer P (2007) Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates. Microbiology 153:281–290

    Article  PubMed  Google Scholar 

  28. Thorpe ED, d’Anjou MC, Daugulis AJ (1999) Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris. Biotechnol Lett 21:669–672

    Article  CAS  Google Scholar 

  29. Xie J, Zhou Q, Du P, Gan R, Ye Q (2005) Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production. Enzym Microb Technol 36:210–316

    Article  CAS  Google Scholar 

  30. Zhu T, Hang H, Chu J, Zhuang Y, Zhang S, Guo M (2013) Transcriptional investigation of the effect of mixed feeding to identify the main cellular stresses on recombinant Pichia pastoris. J Ind Microbiol Biotechnol 40:183–189

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Project N° 11110486 from Fondecyt (Conicyt), Chile.

Author information

Authors and Affiliations

  1. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, Chile

    Julio Berrios, María-Olga Flores, Alvaro Díaz-Barrera, Claudia Altamirano, Irene Martínez & Zaida Cabrera

Authors
  1. Julio Berrios
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María-Olga Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alvaro Díaz-Barrera
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudia Altamirano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Irene Martínez
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zaida Cabrera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Julio Berrios.

Electronic supplementary material

10295_2016_1895_MOESM1_ESM.docx

Supplementary material 1. File contains the nomenclature list as well as the equations involved in the scaling-up procedure.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Berrios, J., Flores, MO., Díaz-Barrera, A. et al. A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation. J Ind Microbiol Biotechnol 44, 407–411 (2017). https://doi.org/10.1007/s10295-016-1895-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-016-1895-7

Keywords

Search

Navigation