Hydrazinoacetic acid is a biosynthetic precursor of the bacterially produced nitramine, N-nitroglycine

Nitramines [R(R')N-NO₂; R,R'=H or alkyl] are valuable synthetic products, but knowledge of the biosynthetic processes that generate these compounds is limited. This work sought to elucidate the biosynthesis of a nitramine natural product, N-nitroglycine (NNG) by Streptomyces noursei. Stable isotope studies showed that S. noursei cells supplemented with l-(ε-¹⁵N)lysine, (¹⁵N)glycine, or (¹³C)hydrazinoacetic acid (HAA) incorporated 67%, 88%, and 67% of the isotope label into NNG, respectively, indicating that these compounds are biosynthetic precursors of NNG. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) of ¹⁵N-Lys-labeled NNG confirmed that the nitro nitrogen of NNG originates from Lys. Bioinformatics analysis of the S. noursei genome showed evidence for a biosynthetic gene cluster (BGC) that contained machinery for HAA biosynthesis (nngKLM), consistent with the results of the isotope labeling. In vitro reconstitution of the gene products produced HAA. The borders of this BGC were defined by cross-referencing the predicted BGC with previously published differential proteomics data. Furthermore, we show that azaserine is produced alongside NNG in S. noursei cultures, linking the two biosynthetic pathways via a proposed nitrosamine biosynthetic intermediate. Finally, the oxygen balance for NNG is -20.2% for the formation of carbon dioxide (CO₂), which is comparable to that of hexahydro-1,3,5-trinitro-1,3,5-triazene (common name: RDX; -21.6%). Crystal structure data of NNG indicate that the unit crystalizes as a pure material, not a hydrate, suggesting a favorable energetic crystallization phase. The combined results suggest a route that, with further development, could lead to sustainable production of energetic nitramines via synthetic biology or biocatalytic approaches.

Chemical manufacture of nitramine energetics produces hazardous waste streams. Mitigation of these waste streams could be accomplished by replacing current production processes with enzymatic or synthetic biology approaches, enabling sustainable production of these compounds. This study identifies a Streptomyces noursei biosynthetic gene cluster that produces a nitramine natural product, N-nitroglycine, via the formation of a hydrazine precursor. The combined data, along with previously reported proteomics data, define the boundaries and genetic inventory of the biosynthetic gene cluster. Furthermore, structural and thermal degradation analyses of the natural product suggest favorable properties for the use of N-nitroglycine, itself, as an energetic material. Further development of this pathway could enable sustainable approaches to produce energetic nitramines either by oxidation of non-native polyhydrazines to the corresponding polynitramines or by improving production and isolation of N-nitroglycine from S. noursei cultures.