Split Intein-Mediated Engineering of Bacteriocins Through Backbone Circularization, Modular Assembly of Chimeras, and Inducible Circularization.

The increasing prevalence of antimicrobial resistance has renewed interest in bacteriocins and other antimicrobial peptides as promising alternatives to conventional antibiotics. In this context, in vitro cell-free protein synthesis (IV-CFPS) provides a rapid and versatile platform for the production and functional evaluation of bacteriocins, enabling antimicrobial screening without the limitations associated with cellular expression, low production yields, or peptide toxicity. In previous studies, we demonstrated the utility of IV-CFPS for the production and characterization of a broad range of bacteriocins. More recently, integration of IV-CFPS with split intein-mediated ligation (SIML) enabled the production and functional evaluation of circular bacteriocins, thereby expanding the repertoire of bacteriocins accessible to cell-free synthesis and screening. Here, we further expand the capabilities of the IV-CFPS/SIML platform through three complementary bacteriocin engineering strategies. First, SIML was applied to the backbone circularization of naturally linear bacteriocins, demonstrating that SIML-mediated circularization can be extended beyond naturally circular bacteriocins. Second, the platform enabled the modular assembly of chimeric bacteriocins through the ligation of the translocation/receptor-binding (T-R) and cytotoxic (C) domains of either the same or different colicins. Finally, we developed a proof-of-concept inducible protein trans-splicing system in which bacteriocin circularization is triggered by the addition of a complementary split intein component, thereby providing temporal control over peptide activation. These results establish IV-CFPS/SIML as a versatile platform for programmable bacteriocin engineering and substantially expand its applications beyond the production of naturally circular bacteriocins.