Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps

Significance Despite significant advances in the prediction of natural product biosynthetic gene clusters (BGCs) from microbial genomes, challenges remain for those belong to the lesser-known classes. Using a bioactivity-guided library screening approach, we have identified a BGC encoding the biosynthesis of anisomycin, an important pyrrolidine-containing protein synthesis inhibitor. The biosynthetic pathway is distinct from known bacterial alkaloid pathways and involves a class of natural product backbone biosynthesis genes encoding an α-keto acid-incorporating transketolase. A cryptic but crucial glycosylation, unexpected transaminations, and a multistep pyrrolidine-forming reaction catalyzed by a single enzyme are also required to complete the assembly of the core benzylpyrrolidine scaffold. These findings open up new avenues for genomics-guided natural product discovery and engineering of pyrrolidine antibiotics.