(28u) Evaluation of Structure-Activity Function of a Lasso Peptide Using High-Throughput Screening

Lasso peptides are natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs). A subset of lasso peptides has targeted antimicrobial activity against pathogenic bacteria. Our group recently discovered and characterized the lasso peptide ubonodin, which has antimicrobial activity against several Burkholderia strains. These bacteria, especially those from the Burkholderia cepacia complex (Bcc), can cause severe lung infections, primarily targeting cystic fibrosis patients. We were interested in cataloging the effects of all possible amino acid substitutions on ubonodin activity as well as identifying variants with enhanced antimicrobial activity. We constructed a library of single mutants and double mutants of ubonodin and developed a next-generation sequencing screening methodology to determine the effect of ubonodin substitutions on the variants’ activity against their target, RNA polymerase. Data from the screen demonstrated that ubonodin tolerates a wide array of substitutions in its loop region. Additionally, the screen revealed an ubonodin variant with improved activity compared to wild-type ubonodin. Overall, the screen of ubonodin variants provides an unprecedented look at the structure-activity relationship of a lasso peptide.