Natural Combinatorial Biosynthesis Involving Two Clusters for the Synthesis of Three Pyrrolamides in Streptomyces netropsis

The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.