Precursor gene engineering expands the loop region of the lasso peptide microcin J25.

Microcin J25 (MccJ25) is the prototypical member of the lasso peptide family and is 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 efforts to generate MccJ25 variants through precursor engineering have largely focused on amino acid substitutions - many of which were poorly tolerated by the biosynthetic machinery and resulted in diminished production. In this work, we demonstrated that the MccJ25 biosynthetic enzymes are surprisingly permissive toward insertions in the loop region. Specifically, the loop can be expanded at different positions by up to 15 additional amino acids and accommodate diverse types of amino acids. Our findings suggest that the lasso synthetase (McjC) has a sizable cavity that tolerates 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.