Heterologous expression and functional optimization of Cecropin A in Escherichia coli Nissle 1917

Cecropin A (CeA) is a cationic antimicrobial peptide with potential antibacterial applications, but its recombinant production remains limited by host toxicity, peptide instability, and low recovery. In this study, an Escherichia coli Nissle 1917-based expression platform was developed for the heterologous production and functional optimization of CeA. A GST-fusion strategy combined with thrombin cleavage and ultrafiltration enabled the recovery of recombinant CeA, and the purified peptide showed antibacterial activity against E. coli JM109, with an MIC of 128 µg/mL. To further improve activity, site-directed and saturation mutagenesis were performed at selected residues. Among the G12 saturation mutants, G12M and G12Y showed the strongest activity, reducing the MIC against E. coli JM109 to 8 µg/mL, corresponding to a 16-fold improvement compared with wild-type CeA. These results indicate that residue 12 is a key position affecting CeA activity and that modulation of local physicochemical properties, especially hydrophobicity, can markedly enhance antibacterial potency. Overall, this study establishes a recombinant production and optimization strategy for CeA and provides useful insights for the engineering of antimicrobial peptides with improved activity.