Abstract
Circumdatins are a unique class of fungal alkaloids featuring a pyrrolo[4,2]benzodiazepine scaffold. In this study, we identified the biosynthetic gene cluster through genome mining guided by structural analysis and docking simulations. Heterologous expression and precursor feeding experiments revealed an unprecedented tailoring pathway in which anthranilate is modified to yield 5-methoxyanthranilate (5-MA), a substrate for nonribosomal peptide synthetase. This work highlights a unique adenylation domain with substrate promiscuity capable of recognizing both anthranilate and 5-MA.