Biosynthesis of a novel benzazepine alkaloid nanangelenin A involves an unprecedented fungal NRPS that incorporates L-kynurenine and catalyzes a regioselective peptide cyclization

Fungi from the genus Aspergillus are prolific producers of bioactive metabolites. Genomic analyses of the biosynthetic gene clusters (BGCs) of Aspergilli, including many surveyed recently, further demonstrate that Aspergillus species are particularly biosynthetically “talented”. Here, we reported the discovery of a novel alkaloid nanangelenin A (1) from a newly-described Australian fungus Aspergillus nanangensis. Structurally, 1 features an unprecedented 3,4-dihydrobenzazepine-2,5-dione-N-prenyl-N-acetoxy-anthranilamide scaffold. We proposed that 1 is biosynthesized via a nonribosomal peptide pathway. Genome analysis identified a putative nonribosomal peptide synthetase (NRPS) gene cluster (nan cluster), which encodes a NRPS (NanA), an acetyltransferase (NanB), an indoleamine- 2,3-dioxyegnase (IDO) (NanC), a prenyltransferase, a methyltransferase (NanE) and a FAD-dependent oxioreductase (NanF). The involvement of nan cluster in the biosynthesis of 1 and the detailed biosynthetic pathway were established by heterologous pathway reconstitution in A. nidulans and substrate feeding. The results proved that NanA incorporates anthranilic acid (Ant) and L-kynurenine (Kyn), and that the unusual benzazepine skeleton in 1 arises from the L-kynurenine, which is converted from an L-tryptophan catalyzed by the indoleamine-2,3-dioxyegnase (IDO) NanC followed by a spontaneous hydrolysis. Using in vivo assays and mutagenesis of NanA, we further demonstrated that the C-terminal condensation and thiolation domains in the NRPS NanA are responsible for the regioselective cyclization of the tethered Ant-Kyn dipeptide, which contains two primary amines. Specifically, the C-terminal condensation domain (C2) catalyzes the regioselective cyclization and the terminal thiolation domain enhances the C2-catalyzed cyclization efficiency.