The most common secondary explosives and propellants contain nitro groups that are produced using chemical nitration reactions. Conventional manufacturing processes for these energetic materials often use hazardous and corrosive substances, such as nitric acid, and produce hazardous waste streams. Highly reactive nitration reactions can also create multiple isomers and by-products that degrade performance of the energetic products. To reduce the environmental impacts of these processes, Strategic Environmental Research and Development Program (SERDP) Statement of Need (SON) WPSON-13-04 requested new synthetic biology strategies to produce energetic chemicals and precursors. This project identified new bionitration mechanisms used by microorganisms to produce nitro-containing natural products, which could be applied to the future production of energetic materials.

The team used systems biology techniques to identify new bionitration enzymes from bacteria to produce nitro compounds. Growth experiments optimized natural product production, and feeding with stable isotope-labeled compounds to infer biosynthetic precursors and proposed pathways. Genome sequencing, quantitative proteomics and bioinformatics analyses will identify proteins that are differentially expressed during periods of peak nitro compound production. Heterologous protein expression, protein purification and in vitro bionitration assays were used to reveal protein function. Determining enzyme mechanisms, specificity and catalytic efficiency established their potential for biosynthetic production of energetic materials.

Five new enzymes were discovered to catalyze 2-aminoimidazole and 2-nitroimidazole (azomycin) biosynthesis in a revised pathway from Streptomyces eurocidicus. Stable isotope labeling experiments identified L-arginine and glycine as precursors for the nitramine N-nitroglycine in Streptomyces noursei. Comparative genomic and proteomic analyses identified a set of proteins that could be responsible for N-nitroglycine biosynthesis. Studies of Salegentibacter sp. demonstrated that nitrate reduction to nitrite was associated with bionitration of phenolic substrates, producing a diverse set of nitrophenols. Gene expression experiments and comparative genome analysis identified two clusters of genes associated with nitrophenol production.

This growing bionitration toolkit represents a diverse range of nitration mechanisms and products that can be adapted for the green chemistry production of nitro compounds and precursors. This work demonstrated feasibility of applying bionitration enzymes to produce relevant nitrated precursors of energetic materials without the use of heavy metal catalysts or strong acids and without producing hazardous waste.