Electric vehicles could cut emissions more than gasoline and hybrid models under varied climates, electricity grids, and battery lifetimes, with the largest gains from light trucks, finds a life-cycle...

Natural products continue to inspire therapeutic innovation due to their structural complexity and biological potency. Pyrrolobenzodiazepines (PBDs), known for antitumor activity, function by covalently binding to guanine bases in DNA, a mechanism that is inherently species-agnostic. However, their potential as antibiotics remains underexplored as modest antibiotic activity is commonly seen with this class of compounds. Here, we produce analogs of tilimycin and tilivalline, two PBDs produced by the gut microbe Klebsiella oxytoca. We mutated NpsA, the nonribosomal peptide synthetase (NRPS) pathway protein responsible for initiating biosynthesis through adenylation of 3-hydroxyanthranilic acid on the pathway to form tilimycin and tilivalline, to enhance promiscuity with substrate analogs. Using structure and informatics-guided mutagenesis, we developed a rapid screening method to identify compatible enzyme-building block combinations to generate a panel of tilimycin and tilivalline analogs. We identified compounds that possess the ability to inhibit DNA polymerase and that show growth inhibitor activity with a DNA-repair mutant of E. coli. This work demonstrates the feasibility of NRPS reprogramming to use biocatalytic approaches to access non-natural derivatives with antibiotic potential and highlights tilimycin analogs as candidates for Gram-negative antibacterial development.