Pseudotetraivprolides from <i>Pseudomonas entomophila</i> Provide Insights into the Biosynthesis of Detoxin/Rimosamide‐Like Anti‐Antibiotics

Novel variants of known natural product (NP) classes can provide valuable insights into their biosynthesis, mechanisms of action, and potential as drug leads across the entire class. Here, we describe a novel member of the widespread detoxine/rimosamide-like (DRL) natural products, named pseudotetraivprolide, produced by Pseudomonas strains. Pseudotetraivprolide exhibits the characteristic DRL-activity of protecting Bacillus cereus against the antibiotic blasticidin S. Through the generation of multiple deletion and complementation mutants, heterologous expression experiments, identification and structure elucidation of several derivatives, chemical synthesis of main derivatives, enzymatic characterization of individual biochemical steps, and detailed homology modelling of enzyme complexes, we elucidated key aspects of its biosynthesis. Our findings demonstrate that the primary metabolism-derived malonyl CoA:ACP transacylase (FabD) functions as a trans-AT in the biosynthesis pathway. Furthermore, we suggest an order for all late-stage modifications and assign a function for the three conserved hypothetical proteins PipDFG acting as last-step acetylation complex responsible for stabilization and activation of the final product.