Mode of action guided metagenomic natural product discovery reveals convergent evolution of a ClpP-targeting motif

The discovery of natural products with specific modes of action from metagenomes remains challenging. Here, we present resistance-CONKAT-seq, a pipeline that links biosynthetic gene clusters (BGCs) to self-resistance genes, enabling identification of metabolites with desired molecular targets. Using clpP-directed resistance-CONKAT-seq, we identify the calprotamides, which activate native ClpP and enhance its activity. Cryo-EM and bioinformatic analyses reveal that the calprotamides' medium-chain N-acylphenylalanine substructure is a convergently evolved ClpP-targeting motif and identify additional BGCs predicted to encode this moiety, including some with co-localized clp genes. The synthesis of structures bioinformatically inspired by two such clp-linked BGCs, desmethyl jomthonic acid C and tuscamide, reveals that both enhance ClpP activity. Extending our bioinformatically guided synthesis study to additional BGCs lacking nearby clp genes shows that ClpP activity enhancement correlated with antibacterial activity, with the strongest enhancers exhibiting narrow-spectrum antibiotic activity. These findings establish N-acylphenylalanine as a previously unrecognized but common natural motif for targeting ClpP, which should help guide the discovery of both natural and synthetic ClpP modulators for antibiotic and anticancer development. Resistance-CONKAT-seq offers a scalable method for exploring biosynthetic dark matter for metabolites with desired modes of action.