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 metagenomic azetidopyrroline (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 IC₅₀ 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.