Discovery and Biosynthesis of Cihunamides, Macrocyclic Antibacterial RiPPs with a Unique C−N Linkage Formed by CYP450 Catalysis

Cihunamides A–D (1–4), novel antibacterial RiPPs, were isolated from volcanic‐island‐derived Streptomyces sp. The structures of 1–4 were elucidated by 1H, 13C, and 15N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C−N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co‐expression of the core genes demonstrated the biosynthesis of cihunamides through P450‐mediated oxidative Trp‐Trp cross‐linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp‐Trp linkage. Cihunamides do not display the non‐canonical atropisomerism shown in tryptorubins, which are the founding members of the “atropitide” family. Therefore, we propose to use a new RiPP family name, “bitryptides”, for cihunamides, tryptorubins, and their congeners, wherein the Trp‐Trp linkages define the structural class rather than non‐canonical atropisomerism. Chemical analysis of volcanic‐island‐derived Streptomyces uncovered cihunamides, unique ribosomally synthesized and post‐translationally modified peptides with antibacterial activity. Genome mining, comparative bioinformatics, and heterologous co‐expression revealed that the key step for cihunamide biosynthesis is the cytochrome P450 mediated oxidative coupling of two tryptophan residues to form an endocyclic C−N bond.