The Isolation of Pyrroloformamide Congeners and Characterization of Their Biosynthetic Gene Cluster.

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. By interfering with zinc ion homeostasis in living cells, they show strong antibacterial activity against a variety of bacterial pathogens, as well as potent cytotoxicity against human cancer cells. In the current study, two new dithiolopyrrolones, pyrroloformamide C () and pyrroloformamide D (), were isolated from sp. CB02980, together with the known pyrroloformamides and . The biosynthetic gene cluster for pyrroloformamides was identified from sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of , which encodes a nonribosomal peptide synthetase (NRPS), abolished the production of -. Overexpression of , a type II thioesterase gene, increased the production of and . Genome neighborhood network analysis of the characterized and orphan gene clusters of dithiolopyrrolones revealed a unified mechanism for their biosynthesis, involving an iterative-acting NRPS and a set of conserved tailoring enzymes for the bicyclic core formation.