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. Darobactins, a recently discovered class of peptide antibiotics, belong to the most promising drug candidates, binding to a new target, the outer membrane protein BamA. Previously, we reported that biosynthetic engineering in a heterologous host allowed for the production of non-natural Darobactins with significantly enhanced antibacterial activity. We here utilize an optimized purification procedure and present cryo-EM structures of the Bam-complex with Darobactin 9 (D9), motivating the biotechnological generation of twenty novel Darobactins including halogenated analogues via structure-based design. 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.