Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

The iron-dependent oxidase UndA cleaves one C3-H bond and the C1-C2 bond of dodecanoic acid to produce 1-undecene and CO₂. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3-H bond using a monoiron cofactor to activate O₂ necessarily invoked unusual or potentially unfeasible steps. Here we present spectroscopic, crystallographic, and biochemical evidence that the cofactor of Pseudomonas fluorescens Pf-5 UndA is actually a diiron cluster and show that binding of the substrate triggers rapid addition of O₂ to the Fe₂(II/II) cofactor to produce a transient peroxo-Fe₂(III/III) intermediate. The observations of a diiron cofactor and substrate-triggered formation of a peroxo-Fe₂(III/III) intermediate suggest a small set of possible mechanisms for O₂, C3-H and C1-C2 activation by UndA; these routes obviate the problematic steps of the earlier hypotheses that invoked a single iron.