Abstract
Plectasin is a promising and potent antimicrobial peptide isolated from the fungus Pseudoplectania nigrella which has been heterologously expressed in various hosts. In this study, a four-copy cassette of plectasin was constructed via 2A peptide assembly to further increase its expression level in recombinant Pichia pastoris. The yeast transformant 4Ple-61 harboring four-copy cassette of plectasin could secrete 183.2 mg/L total protein containing 60.8% of plectasin at the flask level within 120 h, which was 2.3 times higher than that of the yeast transformant Ple-6 carrying one-copy cassette of plectasin. Western blot confirmed the significant peptide expression level in the transformant 4Ple-61. Furthermore, it yielded as high as 426.3 mg/L total protein within 120 h during a 5-L fermentation. The purified plectasin shows superior stability and good antimicrobial activity against conventional Staphylococcus aureus ATCC 26,001 and some food-borne antibiotic-resistant S. aureus strains with the MICs ranging from 8 to 32 μg/mL. Therefore, the strategy based on 2A peptide assembly can enhance the expression of plectasin and further expand its application prospect.
Key points
• A yeast transformant 4Ple-61 with four-copy cassette of plectasin was constructed.
• The plectasin level yield by the transformant 4Ple-61 was boosted by 2.3 times.
• The plectasin showed good activity against food-borne antibiotic-resistant S. aureus.
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Funding
This research was supported by Young Talent of Lifting Engineering for Science and Technology in Shandong Province of China (Grant No. SDAST2021qt18), Taishan Scholar Project of Shandong Province (Grant No. tsqn201812020), and Fundamental Research Funds for the Central Universities (Grant No. 201941002).
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X. L and H. J designed and carried out experiments, analyzed the data, and wrote the manuscript. H. J and X. M reviewed and edited the manuscript, administrated the project, and provided funds. X. S validated the experiments. Q. X, D. M, and P. C performed the literature search and analyzed the data. All authors read and approved the manuscript.
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Liang, X., Jiang, H., Si, X. et al. Boosting expression level of plectasin in recombinant Pichia pastoris via 2A self-processing peptide assembly. Appl Microbiol Biotechnol 106, 3669–3678 (2022). https://doi.org/10.1007/s00253-022-11942-x
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DOI: https://doi.org/10.1007/s00253-022-11942-x