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Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies

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

Abstract

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.

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Acknowledgments

We acknowledge financial support by the National High Technology Research Development Program of P. R. China (863 Program) (Nos. 2014AA093510 and 2011AA02A204), the National Natural Science Foundation of P. R. China (NSFC) (Nos. 31170078, 31070089 and J1103514), the R and D Special Foundation of the ShengZhen Municipal Government (No. JCYJ20120831111657864), the Innovation Foundation of HUST (Nos. 2014QN119 and 2014NY007), and the Fundamental Research Funds for the Central Universities HUST (No. 2172012SHYJ004).

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Authors and Affiliations

  1. Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, People’s Republic of China

    Zhonggang Fang, Li Xu, Dujie Pan, Liangcheng Jiao, Ziming Liu & Yunjun Yan

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  1. Zhonggang Fang
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  2. Li Xu
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  3. Dujie Pan
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Correspondence to Li Xu or Yunjun Yan.

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Fang, Z., Xu, L., Pan, D. et al. Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies. J Ind Microbiol Biotechnol 41, 1541–1551 (2014). https://doi.org/10.1007/s10295-014-1491-7

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  • DOI: https://doi.org/10.1007/s10295-014-1491-7

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