High-Throughput Screen Reveals the Structure–Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin

The Burkholderia
cepacia complex (Bcc)
is a group
of bacteria including opportunistic human pathogens. Immunocompromised
individuals and cystic fibrosis patients are especially vulnerable
to serious infections by these bacteria, motivating the search for
compounds with antimicrobial activity against the Bcc. Ubonodin is
a lasso peptide with promising activity against Bcc species, working
by inhibiting RNA polymerase in susceptible bacteria. We constructed
a library of over 90 000 ubonodin variants with 2 amino acid
substitutions and used a high-throughput screen and next-generation
sequencing to examine the fitness of the entire library, generating
the most comprehensive data set on lasso peptide activity so far.
This screen revealed information regarding the structure–activity
relationship of ubonodin over a large sequence space. Remarkably,
the screen identified one variant with not only improved activity
compared to wild-type ubonodin but also a submicromolar minimum inhibitory
concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants
identified in this study had lower MICs against certain Bcc strains
than those of many clinically approved antibiotics. Finally, the large
library size enabled us to develop DeepLasso, a deep learning model
that can predict the RNAP inhibitory activity of an ubonodin variant.