(427g) Isolation of Functional Variants of the Lasso Antimicrobial Peptide Microcin J25 by High-Throughput Screening and in Silico Design

A lack of antimicrobial drug development combined with emerging multi-drug resistance in recent years demand that new classes of antimicrobials be explored. Microcins are gene-encoded antimicrobial peptides produced by enterobacteria that exert potent activity against closely related bacteria. Microcin J25 (MccJ25) has been of interest to studies due to its unusual lasso structure conferring exceptional stability and its novel mechanism of action ? inhibiting the bacterial RNA polymerase by obstructing the secondary channel. The production of MccJ25 in E. coli is initiated by nutrient depletion during stationary phase, and has been shown to be optimal in minimal media. We have rationally reengineered the gene cluster responsible for the production and maturation/export of the peptide to achieve high production level in rich media. This engineered gene cluster serves as a reliable and high-yield platform for the production of MccJ25 variants. Using both a computational protein design approach and a novel high-throughput screening method for active antimicrobials, we have identified variants of the 21-residue lasso peptide with multiple amino acid substitutions that demonstrate potent antimicrobial activities against Salmonella and E. coli strains. These mutant sequences also provide insight into the structure and activity of the lasso peptide.