Convergent evolution of Streptomyces protease inhibitors involving a tRNA-mediated condensation-minus NRPS

Small peptide aldehydes (SPAs) with protease inhibitory activity are natural products typically synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology, as therapeutic agents, they are physiologically relevant and regulate development of the natural hosts. During genome evolutionary analysis of Streptomyces lividans 66 we identified an NRPS-like biosynthetic gene cluster (BGC) that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA with protease inhibitory activity, called livipeptin. Following genome mining and phylogenomic analyses we confirmed the presence of tRUEs within diverse Streptomyces genomes, including fusions with a C-minus NRPS-like protein. We further demonstrate functional cooperation between these enzymes and provide the biosynthetic rules for the synthesis of livipeptin, expanding the known universe of acetyl-leu/phe-arginal SPAs. The L/F-transferase C-minus NRPS productive interaction was shown to be tRNA-dependent after semisynthetic assays in the presence of RNAse, which contrasts with leupeptin, an acetyl-leu-arginal SPA that we show to be produced by Streptomyces roseous ATCC 31245 via a tRUE-minus BGC with multiple complete NRPSs. Thus, livipeptin and leupeptin are the result of convergent evolution, which has driven the appearance of unprecedented biosynthetic logics directing the synthesis of protease inhibitors thought to be at the core of Streptomyces colony biology. Our results pave the way for understanding this Streptomyces trait, as well as for the discovery of novel natural products following evolutionary genome mining approaches. Abstract importanceConvergent evolution in microbiology is believed to be highly recurrent yet examples that have been comprehensively characterized are scarce. Proteases inhibition by small peptide aldehydes is at the core of many microbiological processes, both within the cell and during colony development, and in microbial ecology. Here we report the biosynthetic foundations of leupeptin, the main Streptomyces protease inhibitor, and of livipeptin, a protease inhibitor produced by Streptomyces lividans. Although these peptides belong to the same chemical class, here we show that their biosynthetic routes result from convergent evolution, as they involve unrelated biosynthetic mechanisms, including the recruitment of a tRNA-utilizing enzyme that functionally replaces the condensation domain of a nonribosomal peptide synthetase during livipeptin biosynthesis. Thus, these results pave the way for understanding Streptomyces protease inhibitors as a trait and provide unprecedented knowledge for genome mining of natural products and synthetic biology where proteases inhibition is desirable.