Resistance-CONKAT-seq Guided Discovery of a ClpP Active Natural Product from a Soil Metagenome

The discovery of natural products with specific molecular targets from metagenomes remains challenging. To address this limitation, we developed resistance-CONKAT-seq (resistance co-occurrence network analysis of targeted sequences) which links metagenomic BGCs (biosynthetic gene clusters) to potential modes of action through the identification of colocalized molecular target-based resistance genes. Applying this approach to a soil metagenomic library, we identified the uncharacterized m e t agenomic a zetidopyrroline (MTA) BGC associated with a potential clpP self-resistance gene. Genetic engineering and heterologous expression of the MTA BGC led to the discovery of metaze A and B, which are structurally related azetidopyrroline- and bicyclocarbamate-based natural products, respectively. Metaze B inhibited Mycobacterium tuberculosis caseinolytic protease proteolytic subunit (ClpP) with an IC 50 of 1.35 μM. This study expands the chemical diversity of natural product ClpP inhibitors and further demonstrates the applicability of resistance-CONKAT-seq for target-guided discovery of natural products with specific modes of action from complex metagenomes.