Abstract
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.
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Data availability
Source Data are provided in the Source Data files with this paper. Demultiplexed reads of clpP genes and ketosynthase (KS) domains are available from Zenodo [https://zenodo.org/records/18763872] (KS) and [https://zenodo.org/records/18763886] (clpP). The assembled sequencing data has been submitted to the NCBI GenBank database under accession no. PV247683. The atomic models have been deposited in the Protein Data Bank under accession codes 9P53 (calprotamide A-bound) and 9P54 (calprotamide A-unbound). The corresponding Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-71299 (calprotamide A-bound) and EMD-71300 (calprotamide A-unbound). The ADEP-bound and apo Mtb ClpP1/P2 structures were obtained from the Protein Data Bank under accession codes 6VGN (ADEP-bound) and 6VGK (apo). Source data are provided with this paper.
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Acknowledgements
We are grateful to Yiming Niu at The Rockefeller University for providing detailed guidance on cryo-EM data analysis. We thank Mark Ebrahim, Johanna Sotiris, Honkit Ng and the Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center for cryo-EM sample preparation and data collection support. This work was supported by National Institutes of Health grant R35GM122559 (SFB). K.S. is supported by NIH T32 GM136640-Tan to K.S.
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S.F.B. conceptualized and supervised the study. J.K. and K.S. designed and performed most experiments and data analysis. A.M. performed the HRMS and NMR data analysis. Y.H. performed the bioinformatics analysis. M.A.T. performed the molecular cloning. J.B. contributed to the design of molecular cloning. C.P. assisted with the large-scale extraction. R.E.B. synthesized tuscamide. A.B. synthesized the jomthonic acid analog. S.F.B., J.K. and K.S. wrote the manuscript.
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Kan, J., Spotton, K., Morales-Amador, A. et al. Mode of action guided metagenomic natural product discovery reveals convergent evolution of a ClpP-targeting motif. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71586-7
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DOI: https://doi.org/10.1038/s41467-026-71586-7
