
Bacterial foodborne contamination poses a dual challenge of chemical preservative risks and antibiotic resistance, drives the need for green production of natural antimicrobial alternatives. The reported cationic antimicrobial peptide (AMP) Spgillcin177−189 derived from the Scylla paramamosain, has strong antimicrobial activity against Staphylococcus aureus and clinical isolation strains. To meet industry demand in future, large-scale production of Spgillcin177− 189 is essential. In the study, Pichia pastoris expression system was established for production of the recombinant Spgillcin177− 189 (rSp gillcin177−189). Then, multicopy strategy was selectively designed by employing the Golden Gate assembly technology to efficiently construct multi-copy plasmid s, which significantly enhanced the expression level of Spgillcin177− 189. A yield of 126.1 mg/L was harvested with 2.75-fold higher that of the single-copy strain. In addition, the recombinant Spgill cin177 − 189 exhibited potent antibacterial activity against multiple foodborne pathogens within a MIC range of 5.25–84 µg/ mL. It also showed effective bactericidal activity and anti-biofilm activity against Staphylococcus aureus and Vibrio parahaemolyticus. rSpgillcin177 − 189 exhibited good thermostability, with no obvious cytotoxicity and hemolytic activity. rSpgillcin177 − 189 may interact with microbial surface components via hydrogen bonding, which were vital for peptide activity in combating bacteria. The rSpgillcin177 − 189 specifically targeting the cell membrane, disrupted bacterial mem brane integrity and leading to cell death. This study provided a very feasible genetic engineering strategy for large-scale production of rSpgillcin177 − 189, which will be applied at a lower cost in agricultural and food industries in future.
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