Rational Engineering and Biosynthesis of Defensin-Derived Antimicrobial Peptides with Broad-Spectrum and Potent Activity

Antibiotic resistance is escalating, highlighting the urgent need for novel antimicrobial strategies. Defensin-like antimicrobial peptides (AMPs) are considered ideal candidates due to their broad-spectrum activity and engineerable potential; however, their limited antimicrobial efficacy and complex chemical synthesis constrain practical applications. In this study, we aimed to enhance the antimicrobial properties of defensin-like AMPs through rational design, directed evolution, and structural fusion strategies. The engineered variant XC1 demonstrated significantly improved antimicrobial activity against a broad range of pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>, while maintaining broad-spectrum efficacy. Comprehensive evaluation of toxicity and stability showed that XC1 exhibited good functional stability in serum, low hemolysis, and low cytotoxicity, indicating excellent therapeutic potential. In addition, high-level secretory expression of defensin-derived AMPs and their engineered variants was achieved using <i>Pichia pastoris</i> GS115, demonstrating strong biosynthetic capability. Together, these results provide a viable strategy for enhancing the antimicrobial activity and scalable biosynthesis of defensin-like AMPs.