Targeted Peptide\nModification Using an Engineered\nBacterial <i>N</i>‑Glycosyltransferase

Many biosynthetic\nenzymes involved in the production\nof ribosomally\nsynthesized and post-translationally modified peptides (RiPPs) in\nprokaryotic organisms often possess two distinct functional parts:\n(1) the RiPP precursor peptide recognition element (RRE) and (2) the\ncatalytic domain. The former binds to the conserved leader portion\nof bipartite precursor peptides, while the latter modifies a specific\nsequence in the core peptide. This makes the enzymes specific yet\npromiscuously act on precursor peptides with diverse core sequences.\nHerein, we engineered fusion enzymes using this biological principle\nby recombinant tethering of a bacterial <i>N</i>-glycosyltransferase\n(NGT) to RREs. Also, we designed guided (with a leader peptide) and\nunguided (no leader peptide) substrates for evaluation of the enzymes.\nThe chimeric system improved the catalytic efficiency as well as the\nin vitro and in vivo selectivity of the fusion enzyme for some guided\nsubstrates compared to the wild-type enzyme. Furthermore, we successfully\ndemonstrated the utility of the engineered enzymes through the production\nof an <i>N</i>-glycosylated analogue of the glycocin sublancin.\nAltogether, this work illustrates the viability of our approach for\ntransforming protein post-translational modification enzymes into\ndesigner tools for introducing new chemical modifications to RiPP\nnatural products and other peptides.