Hyalucidin A: A Bacterial RiPP Featuring a Benzofuranoindoline Motif Biosynthesized by Tandem P450 Catalysis

Abstract Despite the emergence of P450‐modified ribosomally synthesized and post‐translationally modified peptides (RiPPs) as a distinct and rapidly expanding class of natural products, their structural diversity has been limited to scaffolds catalyzed solely by single P450 enzymes. To access greater structural complexity, we targeted unexplored biosynthetic gene clusters encoding two P450s. This effort led to the discovery of hyalucidin A, which features the most intricate polycyclic cross‐linked architecture among reported P450‐RiPPs and represents the first bacterial RiPP containing a benzofuranoindoline motif. Through stepwise enzymatic analysis, combinatorial biosynthesis, and precursor peptide engineering, we deciphered the cooperative function and substrate tolerance of both P450s. Our work expands the biosynthetic logic of P450‐driven cyclization and provides a platform for engineering complex macrocyclic peptides.