Activity and cryo-EM structure guided biosynthetic pathway engineering yields non-natural Darobactin antibiotics with superior activity against Gram-negative pathogens

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated non-natural darobactins with significantly enhanced antibacterial activity. Here we utilise an optimised purification procedure and present cryo-EM structures of the Bam-complex with darobactin 9 (D9), which served as a blueprint to further the biotechnological generation of twenty novel darobactins including halogenated analogues. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, while toxic effects were not observed in human cells and zebrafish embryos up to 500 µg/mL.