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A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains

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Abstract

A battery of Pichia pastoris transformants, G1, G6, A2, A3, C3, carrying 1, 6, 12, 18 and 29 copies of porcine insulin precursor (PIP) gene, were employed to investigate the genetic stability of these multi-copy P. pastoris strains. Both G6 and C3 maintained their original copy numbers in serial culture without methanol induction for 35 generations. With methanol as an inducer and carbon source, G1 and G6 remained stable but the average copy numbers (ACNs) of PIP gene in A2, A3, C3 were decreased to 10, 10 and 15 copies, respectively, after 96 h of induction in shake-flask culture. A PIP copy number distribution analysis of fermentation samples of C3 indicated that the majority of yeast cells have partially or completely lost their PIP genes. In 5-l fermentor culture, the ACNs of PIP gene in A2, A3, C3 were also decreased to 10, 15, 21 copies, respectively, after 72 h of methanol induction.

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References

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

    Article  PubMed  CAS  Google Scholar 

  • Cos O, Ramon 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 Fact 5:17

    Article  PubMed  Google Scholar 

  • Curvers S, Linnemann J, Klauser T, Wandrey C, Takors R (2002) Recombinant protein production with Pichia pastoris in continuous fermentation—kinetic analysis of growth and product formation. Eng Life Sci 8:229–235

    Article  Google Scholar 

  • Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44

    Article  PubMed  Google Scholar 

  • Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  PubMed  CAS  Google Scholar 

  • Harju S, Fedosyuk H, Peterson KR (2004) Rapid isolation of yeast genomic DNA: Bust n’ Grab. BMC Biotechnol 4:8

    Article  PubMed  Google Scholar 

  • Lee FW, Da Silva NA (1997) Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 48:339–345

    Article  PubMed  CAS  Google Scholar 

  • Lim HK, Kim KY, Lee KJ, Park DH, Chung SI, Jung KH (2000) Genetic stability of integrated structural gene of guamerin in recombinant Pichia pastoris. J Microbiol Biotechnol 10:470–475

    CAS  Google Scholar 

  • Luo HY, Huang HQ, Bai YG, Wang YR, Yang PL, Meng K, Yuan TZ, Yao B (2006) Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 22:528–533

    PubMed  CAS  Google Scholar 

  • Mansur M, Cabello C, Hernandez L, Pais J, Varas L, Valdes J, Terrero Y, Hidalgo A, Plana L, Besada V, Garcia L, Lamazares E, Castellanos L, Martinez E (2005) Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol Lett 27:339–345

    Article  PubMed  CAS  Google Scholar 

  • Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R (1998) Chromosomal DNA patterns and gene stability of Pichia pastoris. Yeast 14:895–903

    Article  PubMed  CAS  Google Scholar 

  • Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527–533

    Article  CAS  Google Scholar 

  • Vassileva A, Chugh DA, Swaminathan S, Khanna N (2001) Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. J Biotechnol 21:71–80

    CAS  Google Scholar 

  • Wang X, Wang Z, Da Silva NA (1996) G418 Selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae. Biotechnol Bioeng 49:45–51

    Article  PubMed  CAS  Google Scholar 

  • Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This research was partially supported by grants from National Natural Science Foundation of China (No. 30560006) and High-tech Research Plan of China (863) (2007AA100601). We also thank Dr Hong Hoi Ting (Director of GeneMedix plc, UK) for critical reading of the manuscript.

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Rd, P.O. Box 329#, Shanghai, 200237, People’s Republic of China

    Taicheng Zhu, Meijin Guo, Chen Sun, Jiangchao Qian, Yingping Zhuang, Ju Chu & Siliang Zhang

  2. Department of Biotechnology, Jiangxi Agricultural University, Nanchang, 330045, People’s Republic of China

    Meijin Guo

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  1. Taicheng Zhu
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  2. Meijin Guo
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  3. Chen Sun
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  4. Jiangchao Qian
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  5. Yingping Zhuang
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  6. Ju Chu
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  7. Siliang Zhang
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Correspondence to Meijin Guo.

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Zhu, T., Guo, M., Sun, C. et al. A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett 31, 679–684 (2009). https://doi.org/10.1007/s10529-009-9917-4

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  • DOI: https://doi.org/10.1007/s10529-009-9917-4

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