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Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

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Abstract

In order to improve the extracellular endo-1,4-β-mannosidase (MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides (W1, MF4I, INU1A, αpre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector pGAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4I, INU1A, apre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/mL, while those mediated by MF4I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/mL, respectively. The maximum MAN activity reached 347.5 U/mL with 4 gene copies mediated by MF4I. These results indicate that replacing the signal peptide α-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

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Authors and Affiliations

  1. School of Minerals Processing and Bioengineering, Central South University, Changsha, 410083, China

    Ye Wang  (王冶), Jia Zheng  (郑甲), Fu-lai Lin  (林福来) & Hong-bo Zhou  (周洪波)

  2. Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha, 410083, China

    Hong-bo Zhou  (周洪波)

Authors
  1. Ye Wang  (王冶)
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  2. Jia Zheng  (郑甲)
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  3. Fu-lai Lin  (林福来)
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  4. Hong-bo Zhou  (周洪波)
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Correspondence to Hong-bo Zhou  (周洪波).

Additional information

Foundation item: Project(13JJ9002) supported by Hunan Provincial Natural Science Foundation of China; Project(2012XK4081) supported by the Key Science Technology Plan Project of Hunan Provincial Science & Technology Department, China; Project(CX2012B124) supported by the Graduate Degree Thesis Innovation Program of Hunan Province, China

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Wang, Y., Zheng, J., Lin, Fl. et al. Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide. J. Cent. South Univ. 22, 2088–2095 (2015). https://doi.org/10.1007/s11771-015-2733-1

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  • DOI: https://doi.org/10.1007/s11771-015-2733-1

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