Abstract Darobactin A is a post-translationally modified peptide antibiotic recently isolated from photorhabdus symbionts present in nematodes. The molecule contains a novel pattern of internal oxidative cross links that result in its pre-organization as a beta sheet mimetic. The compound binds to the beta barrel protein BamA and inhibits chaperone functions essential for folding of bacterial outer membrane proteins. Darobactin A is broadly active against gram-negative pathogens in vitro and in animal models of infection. The dar operon encodes a single radical SAM enzyme that catalyzes formation of both oxidative cross links observed in the natural product. The mechanism(s) behind this remarkable outcome is not yet known. We have expressed and purified recombinant His-tagged DarE. When properly reconstituted with iron and sulfur under anaerobic conditions, iron titration data indicates the enzyme contains three iron sulfur clusters and it rapidly generates 5dAdo from SAM – indicating reconstituted DarE is active as a SPASM enzyme. Here we propose a combined chemical synthesis and biosynthesis program to study DarE enzymology and to engineer semi- synthetic forms of the natural product that can be produced on scale. Such studies hold considerable promise. It has been nearly 60 years since a new class of antibiotics active against gram negative infections have been developed.