Decentralized Biosynthesis of the Spiro-DKP Alkaloid Chevalinulin A Enabled by Nonclustered P450 and Intermolecular [4 + 2] Cyclase

Abstract Intermolecular Diels–Alder (DA) cycloadditions are pivotal for constructing complex polycyclic architectures. However, biological catalysts performing this transformation on diketopiperazine (DKP) cores remain largely unidentified. Here, we report a decentralized biosynthetic pathway in the deep-sea cold-seep sourced fungus Aspergillus chevalieri CS-122 that assembles chevalinulin A, a spiro[bicyclo[2.2.2]octane]-DKP alkaloid. This pathway involves two nonclustered enzymes, CalO and FadA. CalO is a cytochrome P450 that furnishes an essential exocyclic olefin, while FadA represents the first FAD oxidoreductase-like Diels–Alderase that catalyzes an intermolecular, endoselective [4 + 2] cycloaddition between neoechinulin B and 3,4-trans-cyclohexadiene. These enzymes function downstream of a conserved gene cluster, effectively decoupling core scaffold assembly from late-stage functionalization. This strategy likely underpins the extensive structural diversity of fungal DKP alkaloids. Notably, CalO and FadA were identified via an integrated transcriptomic and structure-guided computational pipeline, rather than sequence homology, demonstrating the efficacy of this approach in mining functional catalysts from genomic “dark matter”. Mutational analysis indicates that residues M193, Y226, Y315, and D446 are critical in FadA activity. This study reveals a decentralized biosynthetic paradigm linking clustered core assembly with dispersed diversification, establishing a general framework for engineering complex alkaloid scaffolds.