Skip to main content
SpringerLink
Log in

Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03–0.08 h−1 during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h−1. With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.

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
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Bahrami A, Shojaosadati SA, Khalilzadeh R, Vasheghani Farahani E (2008) Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris. Biotechnol Lett 30:1081–1085

    Article  CAS  Google Scholar 

  • Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D (2008) Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 100:177–183

    Article  CAS  Google Scholar 

  • Cao RB, Xu XQ, Zhou B, Chen DS, Chen PY (2004) Gene modification and high prokaryotic expression of porcine interferon alpha-1. Chin J Biotech 20:291–294

    CAS  Google Scholar 

  • Çelik E, Çalık P, Oliver SG (2009) A structured kinetic model for recombinant protein production by Mut+ strain of Pichia pastoris. Chem Eng Sci 64:5028–5035

    Article  Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  Google Scholar 

  • Cereghino GP, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332

    Article  Google Scholar 

  • Chang HW, Jeng CR, Liu JJ, Lin TL, Chang CC, Chia MY, Tsai YC, Pang VF (2005) Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha. Vet Microbiol 108:167–177

    Article  CAS  Google Scholar 

  • Chauhan AK, Arora D, Khanna N (1999) A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochem 34:139–145

    Article  CAS  Google Scholar 

  • Chen X, Xue Q, Zhu R, Fu X, Yang L, Sun L, Liu W (2009) Comparison of antiviral activities of porcine interferon type I and type II. Chin J Biotech 25:806–812

    Google Scholar 

  • Chinsangaram J, Moraes MP, Koster M, Grubman MJ (2003) Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease. J Virol 77:1621–1625

    Article  CAS  Google Scholar 

  • Choi DB, Park EY (2006) Enhanced production of mouse α-amylase by feeding combined nitrogen and carbon sources in fed-batch culture of recombinant Pichia pastoris. Process Biochem 41:390–397

    Article  CAS  Google Scholar 

  • Chun X, Wu D, Wu WX, Wan JQ, Wang L, Yang TY, Wang Q, Ning YB (2005) Cloning and expression of interferon-α/γ from a domestic porcine breed and its effect on classical swine fever virus. Vet Immunol Immunopathol 104:81–89

    Article  Google Scholar 

  • Cos O, Ramon R, Montesinos JL, Valero F (2006) A simple model-based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess. Biotechnol Bioeng 95:145–154

    Article  CAS  Google Scholar 

  • de Avila BS, Brum MC, Bautista E, Koster M, Weiblen R, Golde WT, Grubman MJ (2006) Immunopotentiation of a foot-and-mouth disease virus subunit vaccine by interferon alpha. Vaccine 24:3446–3456

    Article  Google Scholar 

  • Duan SB, Shi ZP, Feng HJ, Duan ZY, Mao ZG (2006) An on-line adaptive control based on DO/pH measurements and ANN pattern recognition model for fed-batch cultivation. Biochem Eng J 30:88–96

    Article  CAS  Google Scholar 

  • Huang H, Xie P, Yu RS, Liu HL, Zhang DF, Cao XR, Li Z (2005) High level secretion expression of pIFN-α in Pichia pastoris. Hereditas 27:215–220

    CAS  Google Scholar 

  • Inan M, Meagher MM (2001) The effect of ethanol and acetate on protein expression in Pichia pastoris. J Biosci Bioeng 92:337–341

    CAS  Google Scholar 

  • 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 37:26–33

    Article  CAS  Google Scholar 

  • Johnson W, Cord-Ruwish R, Cooney MJ (2002) Industrial control of recombinant E. coli fed-batch culture: new perspective on traditional controlled variables. Bioprocess Biosyst Eng 25:111–120

    Article  Google Scholar 

  • Jungo C, Marison I, von Stockar U (2007) Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures. J Biotechnol 130:236–246

    Article  CAS  Google Scholar 

  • Khatri NK, Hoffmann F (2006) Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris. Appl Microbiol Biotechnol 72:492–498

    Article  CAS  Google Scholar 

  • Lee CY, Nakano A, Shiomi N, Lee EK, Katoh S (2003) Effects of substrate feed rates on heterologous protein expression by Pichia pastoris in DO-stat fed-batch fermentation. Enzyme Microb Tech 33:358–365

    Article  CAS  Google Scholar 

  • 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 62:342–348

    Article  CAS  Google Scholar 

  • Liu GS, Lu SY (2004) The immune mechanics of interferon. Chin J Vet Sci Technol 34:72–74

    Google Scholar 

  • Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    Article  CAS  Google Scholar 

  • Min CK, Lee JW, Chung KH, Park HW (2010) Control of specific growth rate to enhance the production of a novel disintegrin, saxatilin, in recombinant Pichia pastoris. J Biosci Bioeng 110:314–319

    Article  CAS  Google Scholar 

  • Ohya T, Ohyama M, Kobayashi K (2005) Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation. Biotechnol Bioeng 90:876–887

    Article  CAS  Google Scholar 

  • Schenk J, Balazs K, Jungo C, Urfer J, Wegmann C, Zocchi A, Marison IW, von Stockar U (2008) Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain. Biotechnol Bioeng 99:368–377

    Article  CAS  Google Scholar 

  • Suye S, Ogawa A, Yokoyama S, Obayashi A (1990) Screening and identification of Candida methanosorbosa as alcohol oxidase-producing methanol using yeast. Agric Biol Chem 54:1297–1298

    Article  CAS  Google Scholar 

  • Wei C, Zhou X, Zhang Y (2008) Improving intracellular production of recombinant protein in Pichia pastoris using an optimized preinduction glycerol-feeding scheme. Appl Microbiol Biotechnol 78:257–264

    Article  CAS  Google Scholar 

  • 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 33:473–483

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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 41:74–78

    Article  Google Scholar 

  • Zhou X, Song Z, Liu X, Jia F, Wang Y (2011) Production of recombinant porcine interferon alpha using PHB-intein-mediated protein purification strategy. Appl Biochem Biotechnol 163:981–993

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the financial supports from the key agricultural technology program of Shanghai Science & Technology Committee (#073919108) and Major State Basic Research Development Program (#2007CB714303) of China as well as Program of Introducing Talents of Discipline to Universities (111-2-06).

Author information

Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Min-Jie Gao, Zhi-Yong Zheng, Jian-Rong Wu, Hu Jin, Xiao-Bei Zhan & Chi-Chung Lin

  2. Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Science, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, 201106, People’s Republic of China

    Shi-Juan Dong & Zhen Li

Authors
  1. Min-Jie Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhi-Yong Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jian-Rong Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shi-Juan Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hu Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiao-Bei Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chi-Chung Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiao-Bei Zhan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gao, MJ., Zheng, ZY., Wu, JR. et al. Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy. Appl Microbiol Biotechnol 93, 1437–1445 (2012). https://doi.org/10.1007/s00253-011-3605-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3605-8

Keywords

Search

Navigation