Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate‐gated chloride channels (GluCls) in invertebrates, not affecting human ligand‐gated anion channels. Okaramines B (1) and D (2) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2‐dimethyl‐3‐methyl‐azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka) of 1 and 2, then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α‐ketoglutarate‐dependent non‐heme FeII dioxygenase that forged the azetidine ring on the okaramine skeleton. Okaramines are a class of complex indole alkaloids and potential insecticidal agents that activate glutamate‐gated chloride channels (GluCls) in invertebrates selectively over human ligand‐gated anion channels. The biosynthesis pathway was mapped with genetic inactivation, pathway enzyme reconstitution in a yeast heterologous system, and biochemical characterization.