Identification of a Nonribosomal Peptide Analog With Activity Against Multiple Gram‐Positive Bacteria via a Synthetic Bioinformatic Natural Product Discovery Approach

ABSTRACT Nonribosomal peptide (NRP) antibiotics exhibit potent biological activities and are broadly used in clinical therapy. Because most microorganisms are difficult to culture and many antibiotic biosynthetic genes are silent, traditional activity tracking approaches face major limitations in the discovery of novel NRPs. Here, based on a synthetic bioinformatic natural product (syn‐BNP) discovery approach that integrates bioinformatics and chemical synthesis, a novel nonribosomal peptide synthetase (NRPS) gene cluster from the genome of Rhodococcus erythropolis D‐1 was mined. A putative NRP scaffold synthesized by the NRPS encoded by this cluster was predicted. Through chemical synthesis and four rounds of structure‐activity relationship (SAR) studies, 37 NRP analogs were ultimately generated. Among these analogs, ZURJC28 shows activity against multiple Gram‐positive bacteria, including two drug‐resistant strains. Mechanistic studies and metabolomics analyses revealed that ZURJC28 exerts membrane‐disruptive activity associated with interaction with phosphatidylglycerol (PG)‐enriched Gram‐positive membranes, leading to membrane damage and widespread metabolic dysregulation. ZURJC28 also shows low cytotoxicity and low hemolytic activity, suggesting its preliminary in vitro safety profile.