Backbone N-methylation is one of the prominent peptide modifications that can greatly enhance the pharmacological properties of a peptide. Naturally occurring backbone N-methylated peptides are produced via nonribosomal or ribosomal pathways, the latter of which was only recently identified in the borosin family of ribosomally synthesized and post-translationally modified peptides. Although previous bioinformatic analyses have revealed new putative genes for borosin biosynthesis, the natural scope of structural and biosynthetic diversity of the borosin family has not been thoroughly explored. Here, we report a comprehensive overview of the borosin family of peptide natural products. Using a genome mining approach, we identified more than 1400 new putative biosynthetic gene clusters for borosins and demonstrated that, unlike those previously reported, most of them are found in bacterial genomes and encode a precursor peptide unfused to its cognate methyltransferase enzyme. Biochemical analysis confirmed the backbone N-methylation of the precursor peptide in trans in eight enzyme–precursor pairs and revealed two novel types of enzyme-recognizing sequences in the precursor peptide. This work significantly expands the biosynthetic diversity of borosins and paves the way for the enzymatic production of diverse backbone N-methylated peptides.
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