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Multiple Copies of the Fusion Gene cflyC-mzfDB3 Enhance the Expression of a Hybrid Antimicrobial Peptide in Pichia pastoris

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Abstract

The codon-optimized zebrafish β-defensin 3 mature peptide gene mzfDB3 and channel catfish c-type lysozyme gene cflyC were used to design the fusion gene cflyC-mzfDB3. In the fusion protein cflyC-mzfDB3, 4×Gly flexible amino acid linker with an enterokinase cleavage site DDDDK was designed to link the mzfDB3 to the C-terminus of cflyC, which potentially contributes to digestion of the recombinant cflyC-mzfDB3 by endogenous enterokinases. The fusion protein was expressed in Pichia pastoris X-33 using expression vectors harboring 1-, 2-, or 4-copies, respectively, of the expression cassette. The cflyC-mzfDB3 gene copy numbers of P. pastoris transformants were quantified using real-time quantitative PCR. Their expression yields showed that the expression level in the 4-copy cflyC-mzfDB3 transformant was 2.67-fold higher than that in the 1-copy cflyC-mzfDB3 transformant, demonstrating that the increase in the cflyC-mzfDB3 gene copy number resulted in increased protein expression. The culture medium supernatant containing recombinant cflyC-mzfDB3 exhibited antibacterial activity against Gram-positive Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, indicating that there may be a synergistic effect of mzfDB3 and cflyC on the peptide’s antibacterial activity. In addition, cell-free P. pastoris medium may be a potential candidate for further development as a natural hybrid antimicrobial solution.

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Funding

This work was supported by grant from the National “13th Five-Year” Key Research and Development Program for Science and Technology Support (grant no. 2016YFD0400106).

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Authors and Affiliations

  1. College of Food Science and Technology, Shanghai Ocean University, 201306, Shanghai, China

    W. Li, Y. Tao, C. F. Song, Y. D. Feng, J. Xie & Y. F. Qian

  2. Shanghai Engineering Research Center of Aquatic-Product Processing and Preservation, 201306, Shanghai, China

    Y. Tao, J. Xie & Y. F. Qian

  3. Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation, Ministry of Agriculture, 201306, Shanghai, China

    Y. Tao, J. Xie & Y. F. Qian

Authors
  1. W. Li
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  2. Y. Tao
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  3. C. F. Song
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Correspondence to Y. Tao.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Li, W., Tao, Y., Song, C.F. et al. Multiple Copies of the Fusion Gene cflyC-mzfDB3 Enhance the Expression of a Hybrid Antimicrobial Peptide in Pichia pastoris . Appl Biochem Microbiol 57, 189–199 (2021). https://doi.org/10.1134/S0003683821020083

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