Genome mining and in vivo characterization of triceptides

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by bacteria. The class defining three-residue cyclophane is installed post-translationally by radical SAM enzymes (triceptide synthases) between the aromatic ring of Ω1 and the Cβ of X3 on Ω1-X2-X3 motifs. Previous studies demonstrated that triceptide synthases are widespread among bacterial genomes and are a promising source of novel peptide natural products and biosynthetic enzymes. Herein, a systematic approach is provided for the characterization of triceptide synthase-mediated cyclophane formation and the associated triceptide natural products. In this method, we utilized a genome-mining approach for the discovery of novel triceptide synthases and the cognate precursor peptides. Using xenorceptide A1 from the xnc biosynthetic gene cluster (BGC) as an example, we provide the experimental method for the in vivo functional validation of the triceptide synthase XncB, production of the natural product in Escherichia coli, and BGC activation in the native bacterial host. In addition, we present how mass spectrometry and nuclear magnetic resonance spectroscopy are used for characterization of triceptides. The protocols described in this chapter will serve as a starting point for the further characterization of triceptide synthases and natural products.