Biosynthesis of a New Benzazepine Alkaloid Nanangelenin A from Aspergillus nanangensis Involves an Unusual l-Kynurenine-Incorporating NRPS Catalyzing Regioselective Lactamization.

1-Benzazepine is a pharmaceutically important scaffold but is rare among natural products. Nanangelenin A ( 1 ), containing an unprecedented 3,4-dihydro-1-benzazepine-2,5-dione- N -prenyl- N -acetoxy-anthranilamide scaffold, was isolated from a novel species of Australian fungus, Aspergillus nanangensis . Genomic and retrobiosynthetic analyses identified a putative nonribosomal peptide synthetase (NRPS) gene cluster ( nan ). The detailed biosynthetic pathway to 1 was established by heterologous pathway reconstitution in A. nidulans , which led to biosynthesis of intermediates nanagelenin B-F ( 2 - 5 and 7 ). We demonstrated that the NRPS NanA incorporates anthranilic acid (Ant) and l-kynurenine (l-Kyn), which is supplied by a dedicated indoleamine-2,3-dioxygenase NanC encoded in the gene cluster. Using heterologous in vivo assays and mutagenesis, we demonstrated that the C-terminal condensation (C _T ) and thiolation (T ₃ ) domains of NanA are responsible for the regioselective cyclization of the tethered Ant-l-Kyn dipeptide to form the unusual benzazepine scaffold in 1 . We also showed that NanA-C _T catalyzes the regioselective cyclization of a surrogate synthetic substrate, Ant-l-Kyn- N -acetylcysteamine, to give the benzazepine scaffold, while spontaneous cyclization of the dipeptide yielded the alternative kinetically favored benzodiazepine scaffold. The discovery of 1 and the characterization of NanA have expanded the chemical and functional diversities of fungal NRPSs.