LAUSR

Modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) boosts their engineering potential for exploring new structures and biological functions. The ω-ester containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages, and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to expand the engineering potential of RiPPs, we present here that the multi-domain architecture of an OEP, plesiocin, can be exploited to create a bifunctional modified peptide. Characterization of plesiocin variants revealed that strong chymotrypsin inhibition relies on the bicyclic structure of the domain in which a leucine residue in the hairpin loop functions as a specificity determinant. Four domains of plesiocin promote simultaneous binding of multiple enzymes, where the C-terminal domain binds chymotrypsin most efficiently. Using this information, we successfully engineered a plesiocin variant in which two different domains inhibit chymotrypsin and trypsin, respectively. This result suggests that the multi-domain architecture of OEPs is a useful platform to engineer multi-functional hybrid RiPPs.