Advertisement

Evaluation of the sub-optimal induction strategies for heterologous proteins production by Pichia pastoris Mut+/MutS strains and related transcriptional and metabolic analysis

  • Luqiang Jia
  • Minjie Gao
  • Jian Yan
  • Shanshan Chen
  • Jiaowen Sun
  • Qiang Hua
  • Jian Ding
  • Zhongping Shi
Original Paper
  • 71 Downloads

Abstract

Heterologous proteins induction by methylotrophic recombinant Pichia pastoris is generally implemented at high cells density condition. Methanol concentration (MeOH) and dissolved oxygen concentration (DO) are two crucial operating parameters controlling proteins production. It is difficult to control MeOH/DO at their desired levels simultaneously due to the extremely high oxygen consumption features. Methanol utilization plus (Mut+) and slow (MutS) strains are the two typical phenotypes of recombinant P. pastoris with quite different dynamic characteristics. Therefore, different MeOH/DO combinational control strategies or sub-optimal induction strategies could be adopted. Environments of “high MeOH/low DO” and “high DO/low MeOH” are the realistic induction strategies. In this study, we summarized our own experimental results (using Mut+/MutS strains to produce human serum albumin-human granulocyte colony stimulating factor—HSA-GCSFm/porcine interferon-α—pIFN-α), and compared to data from the literature using the above mentioned two induction strategies. The results suggested that, heterologous proteins production by Mut+ strains favors “high DO/low MeOH (DO ~ 10%, MeOH ~ 0 g/L)” induction condition, while proteins production by MutS strains prefers “high MeOH/low DO (MeOH 5–10 g/L, DO ~ 0%)” induction environment. Thus, based on the P. pastoris types, the corresponding sub-optimal induction strategies should be applied accordingly. The related metabolic analysis indicating methanol utilizing efficiency and the transcriptional analysis reflecting gene up- or down-regulations involved in several key routes in methanol and sorbitol metabolism were implemented. The analysis results strongly supported the conclusions of using the proposed sub-optimal induction strategies for different heterologous proteins production by Mut+ and MutS strains.

Keywords

P. pastoris Methanol concentration Dissolved oxygen Transcriptome analysis 

Notes

Acknowledgements

The authors thank the financial supports from Natural Science Foundation of China (#21606106), Natural Science Foundation of Jiangsu Province (#BK20150127), the Fundamental Research Funds for the Central Universities (#JUSRP51632A, #JUSRP11536), Industry-Education-Research Cooperation Project of Jiangsu Province (#BY2016022-15), National first-class discipline program of Light Industry Technology and Engineering (#2018-17) and Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Research involving human and animal participants

This article does not contain any studies with human participants performed by any of the authors.

References

  1. Agrawal G, Subramani S (2016) De novo peroxisome biogenesis: evolving concepts and conundrums. Biochim Biophys Acta 5:892–901CrossRefGoogle Scholar
  2. Arnau C, Ramon 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 46:494–500CrossRefPubMedGoogle Scholar
  3. Bonnet C, Espagne E, Zickler D, Boisnard S, Bourdais A, Berteaux-Lecellier V (2006) The peroxisomal import proteins PEX2, PEX5 and PEX7 are differently involved in Podospora anserina sexual cycle. Mol Microbiol 62:157–169CrossRefPubMedGoogle Scholar
  4. Chen SS (2018) High-level expression of human lysozyme in Pichia pastoris. Dissertation, Jiangnan UniversityGoogle Scholar
  5. Damasceno LM, Pla I, Chang HJ, 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 37:18–26CrossRefPubMedGoogle Scholar
  6. Ding J, Gao MJ, Hou GL, Liang KX, Yu RS, Li Z, Shi ZP (2014a) Stabilizing porcine interferon-α production by Pichia pastoris with an ethanol on-line measurement based DO-Stat glycerol feeding strategy. J Chem Technol Biotechnol 89:1948–1953CrossRefGoogle Scholar
  7. Ding J, Zhang CL, Gao MJ, Hou GL, Liang KX, Li CH, Ni JP, Li Z, Shi ZP (2014b) Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy. J Biotechnol 137:35–44CrossRefGoogle Scholar
  8. Englaender JA, Zhu Y, Shirke AN, Lin L, Liu XY, Zhang FM, Gross RA, Koffas MAG, Linhardt RJ (2017) Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris. Appl Microbiol Biotechnol 101:2843–2851CrossRefPubMedGoogle Scholar
  9. Gao MJ, Li Z, Yu RS, Wu JR, Zheng ZY, Shi ZP, Zhan XB, Lin CC (2012) Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift. Bioprocess Biosyst Eng 35:1125–1136CrossRefPubMedGoogle Scholar
  10. Ge L, Li Z, Yu RS, Liu HL, Zhang DF, Zhou ZA, Yin XH (2005) Secretive expression of porcine IFN-α in yeast Pichia pastoris. Chin J Vet Sci 25:289–292Google Scholar
  11. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428CrossRefPubMedPubMedCentralGoogle Scholar
  12. Graifer D, Karpova G (2015) Roles of ribosomal proteins in the functioning of translational machinery of eukaryotes. Biochimie 109:1–17CrossRefPubMedGoogle Scholar
  13. Guan B, Chen FX, Lei JY, Li YH, Duan ZY, Zhu RY, Chen Y, Li HZ, Jin J (2013) Constitutive expression of a rhIL-2-HSA fusion protein in Pichia pastoris using glucose as carbon source. Appl Biochem Biotechnol 171:1792–1804CrossRefPubMedGoogle Scholar
  14. Jia LQ, Tu TY, Huai QQ, Sun JW, Chen SS, Li X, Shi ZP, Ding J (2017) Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency. PLoS ONE 10:e0184602CrossRefGoogle Scholar
  15. Jin S, Ye KM, Shimizu K (1995) Metabolic pathway analysis of recombinant Saccharomyces cerevisiae with a galactose-inducible promoter based on a signal flow modeling approach. J Ferment Bioeng 80:541–551CrossRefGoogle Scholar
  16. Jin H, Zheng ZY, Gao MJ, Duan ZY, Shi ZP, Wang ZX, Jin J (2007) Effective induction of phytase in Pichia pastoris fed-batch culture using an ANN pattern recognition model-based on-line adaptive control strategy. Biochem Eng J 37:26–33CrossRefGoogle Scholar
  17. Jin H, Liu GQ, Ye XF, Duan ZY, Li Z, Shi ZP (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 52:91–98CrossRefGoogle Scholar
  18. Jungo C, Schenk J, Pasquier MM, Marison IW, von Stockar U (2007) A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for production of recombinant avidin with Pichia pastori. J Biotechnol 131:57–66CrossRefPubMedGoogle Scholar
  19. Kim S, Warburton S, Boldogh I, Svensson C, Pon L, d’Anjou M, Stadheim TA, Choi BK (2013) Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris. J Biotechnol 166:174–181CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kupcsulik B, Sevella B, Ballagi A, Kozma J (2001) Evaluation of three methanol feed strategies for recombinant Pichia pastoris MutS fermentation. Acta Aliment 30:99–111CrossRefGoogle Scholar
  21. Lee CY, Lee SJ, Jung KH, Katoh S, Lee EK (2003) High dissolved oxygen tension enhances heterologous protein expression by recombinant Pichia pastoris. Process Biochem 38:1147–1154CrossRefGoogle Scholar
  22. Lim HK, Choi SJ, Kim KY, Jung KH (2003) Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch. Appl Microbiol Biotechnol 62:342–348CrossRefPubMedGoogle Scholar
  23. Lu H, Zhu YF, Xiong J, Wang R, Jia ZP (2015) Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae. Microbiol Res 177:28–33CrossRefPubMedGoogle Scholar
  24. Mattanovich D, Callewaert N, Rouzé P (2009) Open access to sequence: browsing the Pichia pastoris genome. Microb Cell Fact 8:1–4CrossRefGoogle Scholar
  25. Mayson BE, Kilburn DG, Zamost BL, Raymond CK, Lesnicki GJ (2003) Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica. Biotechnol Bioeng 81:291–298CrossRefPubMedGoogle Scholar
  26. Ponte X, Luis Montesinos-Segui J, Valero F (2016) Bioprocess efficiency in Rhizopus oryzae lipase production by Pichia pastoris under the control of PAOX1 is oxygen tension dependent. Process Biochem 51:1954–1963CrossRefGoogle Scholar
  27. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842CrossRefPubMedPubMedCentralGoogle Scholar
  28. Smith JJ, Aitchison JD (2013) Peroxisomes take shape. Nat Rev Mol Cell Biol 14:803–817CrossRefPubMedPubMedCentralGoogle Scholar
  29. Tan ZB, Li JF, Wu MC, Tang CD, Zhang HM, Wang JQ (2011) High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris. World J Microbiol Biotechnol 27:2767–2774CrossRefGoogle Scholar
  30. Theron CW, Berrios J, Delvigne F, Fickers P (2018) Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris. Appl Microbiol Biotechnol 102:63–80CrossRefPubMedGoogle Scholar
  31. Wang XD, Jiang T, Yu XW, Xu Y (2017) Effects of UPR and ERAD pathway on the prolyl endopeptidase production in Pichia pastoris by controlling of nitrogen source. J Ind Microbiol Biotechnol 44:1053–1063CrossRefPubMedGoogle Scholar
  32. Wu D, Yu XW, Wang TC, Wang R, Xu Y (2011) High yield Rhizopus chinenisis prolipase production in Pichia pastoris: impact of methanol concentration. Biotechnol Bioprocess Eng 16:305–311CrossRefGoogle Scholar
  33. Yu RS, Dong SJ, Zhu YM, Jin H, Gao MJ, Duan ZY, Zheng ZY, Shi ZP, Li Z (2010) Effective and stable porcine interferon-α production by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis. Bioprocess Biosyst Eng 33:473–483CrossRefPubMedGoogle Scholar
  34. Zhang WH, 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 70:1–8CrossRefPubMedGoogle Scholar
  35. Zhang JG, Wang XD, Su EZ, Fang GC, Ren YH, Wei DZ (2008) A new fermentation strategy for S-adenosylmethionine production in recombinant Pichia pastoris. Biochem Eng J 41:74–78CrossRefGoogle Scholar
  36. Zhu SF, Zhang LF, Chen Y, Chu M, Li Y, Jin J (2008) Expression of mutant HSA-hGCSF in Pichia pastoris. Chin J Biochem Pharm 29:289–293Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Luqiang Jia
    • 1
  • Minjie Gao
    • 1
  • Jian Yan
    • 1
  • Shanshan Chen
    • 1
  • Jiaowen Sun
    • 1
  • Qiang Hua
    • 2
  • Jian Ding
    • 1
  • Zhongping Shi
    • 1
  1. 1.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of BiotechnologyMinistry of Education, Jiangnan UniversityWuxiChina
  2. 2.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina

Personalised recommendations