Biosynthesis of ent-acu-dioxomorpholine A and its conversion to prenyl-rearranged and rare ketooxadiazepane products in heterologous host

Diketomorpholines (DKMs) represent an intriguing class of biologically active heterocycles. To identify unusual DKM natural products, we conducted a comprehensive analysis of fungal genomes from NCBI and our laboratory databases. A non-ribosomal peptide synthetase (NRPS) biosynthetic gene cluster (BGC) was identified in Aspergillus aculeatus CRI323-04, which also harbors a dimethylallyl tryptophan synthase (DMATS) gene and an NAD(P)H-dependent reductase gene. Heterologous expression of this BGC in chassis organism A. nidulans resulted in the production of prenylated DKM ( 3 ), seco -diketomorpholines ( seco -DKMs) with regular ( 6 – 7 ) or reverse prenyl group ( 8 ), as well as rare ketooxadiazepane alkaloids ( 4 – 5 ). Through integrated in vivo and in vitro experiments, we elucidated the biosynthetic pathway of the prenylated DKM ( 3 ). Moreover, the feeding experiment showed that the complex conversion of prenylated DKM ( 3 ) to seco -DKMs ( 6 – 8 ) and ketooxadiazepane ( 4 – 5 ) alkaloids may be mediated by heterologous host A. nidulans . This study elucidates the biosynthetic pathway of 3 and further facilitates the exploration of structurally diverse prenylated DKMs and ketooxadiazepane alkaloids from fungal resources.