Biosynthesis- and Metabolomics-guided discovery of antimicrobial cyclopeptides against drug-resistant clinical isolates

Antimicrobial resistance remains a significant global threat, contributing significantly to mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides (RiPPs) have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we reported the discovery of new Avi(Me)Cys-containing cyclopeptide antibiotics through a synergistic approach that combines rule-based genome mining, automated metabolomic analysis, and heterologous expression. We first bioinformatically identified 1,172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing cyclopeptides from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully established the connection between three newly identified BGCs and the synthesis of five new peptide antibiotics. Notably, massatide A displayed excellent activity against a spectrum of gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus , with a minimum inhibitory concentration (MIC) of 0.25 μg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing cyclopeptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.