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 (3) and pyrroloformamide D (4), were isolated from Streptomyces sp. CB02980, together with the known pyrroloformamides 1 and 2. The biosynthetic gene cluster for pyrroloformamides was identified from Streptomyces sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a nonribosomal peptide synthetase (NRPS), abolished the production of 1-4. Overexpression of pyfN, a type II thioesterase gene, increased the production of 1 and 2. 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.