Targeted Peptide Modification Using an Engineered Bacterial <i>N</i>-Glycosyltransferase

Many biosynthetic enzymes involved in the production of ribosomally synthesized and post-translationally modified peptides (RiPPs) in prokaryotic organisms often possess two distinct functional parts: (1) the RiPP precursor peptide recognition element (RRE) and (2) the catalytic domain. The former binds to the conserved leader portion of bipartite precursor peptides, while the latter modifies a specific sequence in the core peptide. This makes the enzymes specific yet promiscuously act on precursor peptides with diverse core sequences. Herein, we engineered fusion enzymes using this biological principle by recombinant tethering of a bacterial N-glycosyltransferase (NGT) to RREs. Also, we designed guided (with a leader peptide) and unguided (no leader peptide) substrates for evaluation of the enzymes. The chimeric system improved the catalytic efficiency as well as the in vitro and in vivo selectivity of the fusion enzyme for some guided substrates compared to the wild-type enzyme. Furthermore, we successfully demonstrated the utility of the engineered enzymes through the production of an N-glycosylated analogue of the glycocin sublancin. Altogether, this work illustrates the viability of our approach for transforming protein post-translational modification enzymes into designer tools for introducing new chemical modifications to RiPP natural products and other peptides.