Using genome mining and heterologous expression, we report the discovery and production of a new antimicrobial lasso peptide from species related to the Enterobacter cloacae complex. Using NMR and mass spectrometric analysis, we show that this lasso peptide, named cloacaenodin, employs a threaded lasso fold which imparts proteolytic resistance that its unthreaded counterpart lacks. Cloacaenodin has selective, low micromolar, antimicrobial activity against species related to the E. cloacae complex, including species implicated in nosocomial infections and against clinical isolates of carbapenem-resistant Enterobacterales. We further used site-directed mutagenesis to probe the importance of specific residues to the peptide's biosynthesis, stability, and bioactivity.

Through genome mining efforts, we discovered two lasso peptide biosynthetic gene clusters (BGCs) within two different species of Achromobacter, a genus that contains pathogenic organisms that can infect patients with cystic fibrosis. Using gene-refactored BGCs in E. coli, we heterologously expressed two lasso peptides, which we named achromonodin-1 and achromonodin-2. Achromonodin-1 is naturally encoded by certain isolates from the sputum of patients with cystic fibrosis. We solve the NMR structure of achromonodin-1, demonstrating that it is a threaded lasso peptide with a large loop and short tail structure, reminiscent of previously characterized lasso peptides that inhibit RNA polymerase (RNAP). We then show that achromonodin-1 inhibits RNAP in vitro and has potent but narrow-spectrum activity towards Achromobacter pulmonis, another isolate from the sputum of a cystic fibrosis patient. Our efforts expand the repertoire of antimicrobial lasso peptides and provide insights into how Achromobacter isolates from certain ecological niches may interact with each other.