Heterologous Characterization of Mechercharmycin a Biosynthesis Reveals Alternative Insights into Post-Translational Modifications for Ribosomal Peptides

Mechercharmycin (MCM) A is a marine natural product belonging to a family of polyazole cyclopeptides with remarkably biological activity and fascinating structure. Identification, comparative analysis and heterologous expression of the biosynthetic gene cluster revealed a ribosomally synthesized and post-translationally modified peptide (RiPP) system possessing complex and distinctive post-translational modification steps. Combinatorial co-production of precursor peptide with different modified enzymes in Escherichia coli and Bacillus subtilis discovered a different timing of modifications. We show that a tRNAGlu-dependent highly regioselective dehydration is the first modification step, then polyazoles were formed through cyclization and dehydrogenation in a N-to-C terminal direction. In addition, two MCM analogues with comparable antitumor activity were generated by engineering the pathway. This study thereby defined a new subfamily of azol(in)e-containing RiPPs, which set the stage for the next mechanism investigations and combinatorial biosynthesis.