This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

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.Nitrogen-nitrogen (NÀ N) bond-containing functional groups are prominent in small-molecule drugs and enabling chemical reagents. [1] More rarely, these structural motifs are also found in microbial natural products, including secondary metabolites containing diazo, hydrazone, hydrazine, hydrazide, azo, azine, azoxy, N-nitroso, and N-hydroxytriazene groups. [2] Recent efforts have begun to reveal the biosynthetic enzymes involved in assembling these functional groups, enabling both the discovery of new natural products through genome mining and the elucidation of enzymatic strategies for accessing NÀ N bond-containing molecules. [3] Diazo-containing molecules are particularly interesting due to their synthetic utility and application in bioorthogonal transformations. [4] Diazo compounds are powerful enabling reagents for CÀ H and XÀ H insertion reactions (X=N, O, S, P, Si), cyclopropanation, alkylation, ring expansion, and dipolar cycloaddition reactions. [1b, 5] In particular, α-diazoesters, such as ethyl diazoacetate, have recently seen extensive use as substrates for engineered hemedependent enzymes that perform stereoselective carbenemediated reactions. [6] Diazo compounds are also used in bioorthogonal cycloaddition reactions with strained alkynes, offering improved yields and kinetics compared to analogous azide coupling partners under certain conditions. [7] Though relatively few diazo-containing natural products have been reported (Figure 1a), a large proportion of these compounds exhibit cytotoxic activity. [4] For example, the bioactivity of the diazofluorene antitumor antibiotics kinamycin A-D and lomaiviticin A derives from DNA damage inflicted by the reactive diazo warhead(s). [8] Little is known about the biosynthesis of diazo groups, with only four diazoforming enzymes discovered to date.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

No abstract

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.Nitrogen-nitrogen (NÀ N) bond-containing functional groups are prominent in small-molecule drugs and enabling chemical reagents. [1] More rarely, these structural motifs are also found in microbial natural products, including secondary metabolites containing diazo, hydrazone, hydrazine, hydrazide, azo, azine, azoxy, N-nitroso, and N-hydroxytriazene groups. [2] Recent efforts have begun to reveal the biosynthetic enzymes involved in assembling these functional groups, enabling both the discovery of new natural products through genome mining and the elucidation of enzymatic strategies for accessing NÀ N bond-containing molecules. [3] Diazo-containing molecules are particularly interesting due to their synthetic utility and application in bioorthogonal transformations. [4] Diazo compounds are powerful enabling reagents for CÀ H and XÀ H insertion reactions (X=N, O, S, P, Si), cyclopropanation, alkylation, ring expansion, and dipolar cycloaddition reactions. [1b, 5] In particular, α-diazoesters, such as ethyl diazoacetate, have recently seen extensive use as substrates for engineered hemedependent enzymes that perform stereoselective carbenemediated reactions. [6] Diazo compounds are also used in bioorthogonal cycloaddition reactions with strained alkynes, offering improved yields and kinetics compared to analogous azide coupling partners under certain conditions. [7] Though relatively few diazo-containing natural products have been reported (Figure 1a), a large proportion of these compounds exhibit cytotoxic activity. [4] For example, the bioactivity of the diazofluorene antitumor antibiotics kinamycin A-D and lomaiviticin A derives from DNA damage inflicted by the reactive diazo warhead(s). [8] Little is known about the biosynthesis of diazo groups, with only four diazoforming enzymes discovered to date.