Precursor Gene Engineering Expands the Loop Region of the Lasso Peptide Microcin J25

Abstract

Microcin J25 ( MccJ25) is the prototypical member of the lasso peptide family, characterized by a mechanically interlocked structure. Scientists have long been interested in MccJ25 for its potent RNA polymerase inhibitory activity and unique threaded topology. Previous attempts to generate MccJ25 variants by precursor engineering have largely focused on substitutions – many of which were poorly tolerated by the biosynthetic machinery and resulted in diminished production. In this manuscript, we demonstrated that the MccJ25 biosynthetic enzymes are surprisingly permissive toward insertions in the loop region, Specifically, it can be expanded at different positions, by up to 15 additional amino acids, and with diverse types of amino acids. Our findings imply that the lasso synthetase (McjC) has a sizable cavity to accommodate loop expansions. More broadly, this work establishes a new strategy for constructing mechanically interlocked molecules and potentially enables the grafting of diverse bioactive ligands onto the MccJ25 scaffold.