Biotechnology and Bioprocess Engineering

, Volume 22, Issue 6, pp 767–773 | Cite as

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

  • Li Chen
  • Ali Mohsin
  • Ju Chu
  • Yingping Zhuang
  • Yamei Liu
  • Meijin Guo
Research Paper


Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.


sorbitol co-feeding multi-copy Pichia pastoris porcine insulin precursor 


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  1. 1.
    Kim, M. J., S. H. Kim, J. H. Lee, J. H. Seo, J. H. Kim, Y. H. Kim, and S. W. Nam (2008) High-level secretory expression of human procarboxypeptidase B by fed-batch cultivation of Pichia pastoris and its partial characterization. J. Microbiol. Biotechnol. 18: 1938–1944.PubMedGoogle Scholar
  2. 2.
    Adivitiya, V. K. Dagar, N. Devi, and Y. P. Khasa (2016) High level production of active streptokinase in Pichia pastoris fedbatch culture. Int. J. Biol. Macromol. 83: 50–60.CrossRefPubMedGoogle Scholar
  3. 3.
    Cregg, J. M., J. L. Cereghino, J. Shi, and D. R. Higgins (2000) Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16: 23–52.CrossRefPubMedGoogle Scholar
  4. 4.
    Weidner, M., M. Taupp, and S. J. Hallam (2010) Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. J. Vis. Exp. 36: 1862.Google Scholar
  5. 5.
    Qin, X., J. Qian, G. Yao, Y. Zhuang, S. Zhang, and J. Chu (2011) GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl. Environ. Microbiol. 77: 3600–3608.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Capone, S., J. Horvat, C. Herwig, and O. Spadiut (2015) Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter. Microb. Cell Fact. 14: 101.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Scorer, C. A., J. J. Clare, W. R. McCombie, M. A. Romanos, and K. Sreekrishna (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology. 12: 181–184.PubMedGoogle Scholar
  8. 8.
    Shen, Q., M. Wu, H. B. Wang, H. Naranmandura, and S. Q. Chen (2012) The effect of gene copy number and co-expression of chaperon on production of albumin fusion proteins in Pichia pastoris. Appl. Microbiol. Biotechnol. 96: 763–772.CrossRefPubMedGoogle Scholar
  9. 9.
    Charoenrat, T., K. Sangprapai, P. Promdonkoy, K. Kocharin, S. Tanapongpipat, and N. Roongsawang (2015) Enhancement of thermostable β-glucosidase production in a slow methanol utilization strain of Pichia pastoris by optimization of the specific methanol supply rate. Biotechnol. Bioproc. Eng. 20: 315–323.CrossRefGoogle Scholar
  10. 10.
    Looser, V., B. Bruhlmann, F. Bumbak, C. Stenger, M. Costa, A. Camattari, D. Fotiadis, and K. Kovar (2015) Cultivation strategies to enhance productivity of Pichia pastoris: A review. Biotechnol. Adv. 33: 1177–1193.CrossRefPubMedGoogle Scholar
  11. 11.
    Macauley-Patrick, S., M. L. Fazenda, B. McNeil, and L. M. Harvey (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast. 22: 249–270.CrossRefPubMedGoogle Scholar
  12. 12.
    Minning, S., A. Serrano, P. Ferrer, C. Sola, R. D. Schmid, and F. Valero (2001) Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J. Biotechnol. 86: 59–70.CrossRefPubMedGoogle Scholar
  13. 13.
    Mansur, M., C. Cabello, L. Hernandez, J. Pais, L. Varas, J. Valdes, Y. Terrero, A. Hidalgo, L. Plana, V. Besada, L. Garcia, E. Lamazares, L. Castellanos, and E. Martinez (2005) Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol. Lett. 27: 339–345.CrossRefPubMedGoogle Scholar
  14. 14.
    Mattanovich, D., B. Gasser, H. Hohenblum, and M. Sauer (2004) Stress in recombinant protein producing yeasts. J. Biotechnol. 113: 121–135.CrossRefPubMedGoogle Scholar
  15. 15.
    Zhu, T. C., M. J. Guo, C. Sun, J. C. Qian, Y. P. Zhuang, J. Chu, and S. L. Zhang (2009) A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol. Lett. 31: 679–684.CrossRefPubMedGoogle Scholar
  16. 16.
    Zhu, T. C., M. J. Guo, Y. P. Zhuang, J. Chu, and S. L. Zhang (2011) Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes. Appl. Microbiol. Biotechnol. 89: 1127–1135.CrossRefPubMedGoogle Scholar
  17. 17.
    Prabhu, A. A., B. Mandal, and V. V. Dasu (2017) Medium optimization for high yield production of extracellular human interferon-γ from Pichia pastoris: A statistical optimization and neural network-based approach. Kor. J. Chem. Eng. 1–13.Google Scholar
  18. 18.
    Arnau, C., R. Ramon, P. Ferrer, and F. Valero (2007) Sorbitol cofeeding an efficient strategy to reduce metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. J. Biotechnol. 131: S76–S76.CrossRefGoogle Scholar
  19. 19.
    Calik, P., E. Celik, and S. G. Oliver (2009) Recombinant protein production by Mut+ strain of Pichia pastoris using dual carbon sources: methanol and sorbitol. New Biotechnol. 25: S60–S60.CrossRefGoogle Scholar
  20. 20.
    Prabhu, A. A., V. D. Veeranki, and S. J. Dsilva (2016) Improving the production of human interferon gamma (hIFN-γ) in Pichia pastoris cell factory: An approach of cell level. Proc. Biochem. 51: 709–718.CrossRefGoogle Scholar
  21. 21.
    Zhu, T., H. Hang, J. Chu, Y. Zhuang, S. Zhang, and M. Guo (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.CrossRefPubMedGoogle Scholar
  22. 22.
    Athmaram, T. N., S. Saraswat, A. K. Singh, M. K. Rao, N. Gopalan, V. V. Suryanarayana, and P. V. Rao (2012) Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris. Virus Genes. 45: 440–451.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhu, T., L. You, F. Gong, M. Xie, Y. Xue, Y. Li, and Y. Ma (2011) Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline betamannanase in Pichia pastoris. Enz. Microb. Technol. 49: 407–412.CrossRefGoogle Scholar
  24. 24.
    Zhu, T., M. Guo, Z. Tang, M. Zhang, Y. Zhuang, J. Chu, and S. Zhang (2009) Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris. J. Appl. Microbiol. 107: 954–963.CrossRefPubMedGoogle Scholar
  25. 25.
    Hohenblum, H., N. Borth, and D. Mattanovich (2003) Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J. Biotechnol. 102: 281–290.CrossRefPubMedGoogle Scholar
  26. 26.
    Camara, E., J. Albiol, and P. Ferrer (2016) Droplet digital PCRaided screening and characterization of Pichia pastoris multiple gene copy strains. Biotechnol. Bioeng. 113: 1542–1551CrossRefPubMedGoogle Scholar
  27. 27.
    Pazhang, M., F. Mehrnejad, Y. Pazhang, H. Falahati, and N. Chaparzadeh (2016) Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents. Biotechnol. Appl. Biochem. 63: 206–213.CrossRefPubMedGoogle Scholar
  28. 28.
    Sreekrishna, K., R. G. Brankamp, K. E. Kropp, D. T. Blankenship, J. T. Tsay, P. L. Smith, J. D. Wierschke, A. Subramaniam, and L. A. Birkenberger (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene. 190: 55–62.CrossRefPubMedGoogle Scholar
  29. 29.
    Ding, J., C. Zhang, M. Gao, G. Hou, K. Liang, C. Li, J. Ni, Z. Li, and Z. Shi (2014) Enhanced porcine circovirus cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy. J. Biotechnol. 177: 35–44.CrossRefPubMedGoogle Scholar
  30. 30.
    Calik, P., B. Bozkurt, G. Zerze, B. Inankur, E. Bayraktar, E. Boy, M. A. Orman, E. Acik, and T. Ozdamar (2013) Effect of cosubstrate sorbitol different feeding strategies on human growth hormone production by recombinant Pichia pastoris. J. Chem. Technol. Biotechnol. 88: 1631–1640.CrossRefGoogle Scholar
  31. 31.
    Arnau, C., R. Ramon, C. Casas, and F. Valero (2010) Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fedbatch bioprocess. Enz. Microb. Technol. 46: 494–500.CrossRefGoogle Scholar
  32. 32.
    Jungo, C., J. Schenk, M. Pasquier, I. W. Marison, and U. von Stockar (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.CrossRefPubMedGoogle Scholar
  33. 33.
    Xie, J., Q. Zhou, P. Du, R. Gan, and Q. Ye (2005) Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production. Enz. Microb. Technol. 36: 210–216.CrossRefGoogle Scholar
  34. 34.
    Wang, Z. H., Y. Wang, D. X. Zhang, J. H. Li, Z. Z. Hua, G. C. Do, and J. Chen (2010) Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour. Technol. 101: 1318–1323.CrossRefPubMedGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Li Chen
    • 1
    • 2
  • Ali Mohsin
    • 2
  • Ju Chu
    • 2
  • Yingping Zhuang
    • 2
  • Yamei Liu
    • 1
    • 3
  • Meijin Guo
    • 2
  1. 1.National Research Center of Engineering and Technology for Veterinary BiologicalsJiangsu Academy of Agricultural SciencesNanjingChina
  2. 2.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina
  3. 3.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and ZoonosesYangzhouChina

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