Nitroorganic compounds are widely used as energetic materials in propellants and explosives. They are currently produced through chemical synthesis. The nitroorganic explosives and propellants (energetic compounds) utilized in modern conventional munitions are currently synthesized in chemical processes using primarily petroleum-derived feedstocks for the carbon skeleton. Biotechnological routes to the energetic compounds and/or their precursors would utilize plant biomass-derived sugars and simple nitrogen compounds without the use of strong acids, large amounts of organic solvents, or heavy metal catalysts. To decrease environmental impact, this project explored the biosynthetic route to synthesize nitroorganics. Biosynthetic pathways would have a reduced environmental impact and liberate the production of these energetic materials from the need for any foreign-sourced materials or catalysts. To biosynthesize nitroorganics, the genes encoding the enzymes that comprise the pathways to various types of nitroorganic compounds must first be identified and then recombined by synthetic biology techniques to obtain new biosynthetic routes to nitroorganic compounds of interest.

The principal aim in this project was to populate the synthetic biology toolbox with well characterized genes for the biosynthesis of nitroorganic compounds.

The project team accomplished the project goals by 1) Identifying candidate fungi that produce nitroorganic compounds and then further characterizing the fungi and their nitroorganic product profile. 2) Performing comparative transcriptomic studies of the fungi to identify the biosynthetic genes and pathways responsible for producing these nitroorganics. 3) Cloning and expression of candidate genes to demonstrate their biosynthetic utility and to allow characterization of the nitroorganic products profile of the enzymes.

The project team identified candidate fungi that produce representatives of bioorganic compounds, A. oryzae for producing 3-nitropropionic acid (3-NPA), P. citrinum for producing Citrinalin, and A. wentii for producing 1-amino-2-nitro cyclopentane carboxylic acid (ANCPA). A list of oxidoreductase class gene candidates for 3-NPA production was identified from an Aspergillus oryzae strain by comparative transcriptomics of samples taken from a time course of 3-NPA production grown under two culture conditions that result in gram per liter quantities of 3-NPA, or no 3-NPA. In P. citrinum, citrinalin biosynthesis is driven by a secondary metabolite gene cluster containing a non-ribosomal peptide synthase (NRPS). citG was demonstrated to be essential to produce the nitro group by clustered regularly interspaced short palindromic repeats. The A. wentii genome was sequenced to enable the development of genetic engineering tools for identifying genes involved in ANCPA biosynthesis.

This project provided foundational research that will begin to fill the synthetic biology toolbox of "relevant and new biocatalysts" (i.e., enzymes and the genes that encode them) in the production of nitroorganics without the use of solvents, metal catalysts, or strong acids and utilize sugars as carbon sources instead of petroleum – in contrast to existing chemical processes.