New chemical synthesis procedures that are more environmentally friendly for existing or novel energetics is currently being sought by the Department of Defense (DoD). Synthetic processes for nitro compound production traditionally involve concentrated sulfuric and nitric acids, exhibit poor regioselectivity and functional group tolerance, and generate nitrogen oxides and super stoichiometric quantities of acidic waste. Instead, naturally occurring non-heme diiron N-monooxygenases are enzymes that can selectively oxidize aromatic amines to their associated nitro compounds using iron-catalyzed activation of molecular oxygen under mild and ambient conditions with no associated chemical byproducts. While the promise of N-monooxygenases for energetics synthesis is high, their applicability to industrial biotechnology is unknown given that their substrate specificity has undergone limited investigation, their activity outside of cells is impaired, and their activity in cells can be counteracted by multiple redundant nitroreductases that are native to the cell. Here, the objectives are to harness the expertise in synthetic biology to create bacterial strains in which aromatic and heterocyclic nitro compounds are stabilized, to design genetically encoded biosensors for detection of nitro compounds in high-throughput, and to create a cell-based screen to ascertain the molecular basis of N-oxygenase substrate specificity and activity.

It is hypothesized that three key innovations will enable the project team to advance the frontier of knowledge regarding nitro compound biosynthesis. First is the use of advanced multiplexed genome engineering technology to rapidly and combinatorially delete native genes that encode nitroreductases in E. coli, with which has been seen preliminary success in stabilizing many compounds that are otherwise rapidly degraded by the cell. Using this technology, the project team will eliminate all known or putative nitroreductase enzymes from the cell and then measure the stability of diverse aromatic and heterocyclic nitro compounds. Second is the advanced understanding of non-heme diiron N-monooxygenases, which is aided by a collection of 20+ natural N-monooxygenase variants and has resulted in robust activity using previously unreported nonheme diiron N-monooxygenases. These N-monooxygenases and other similar sequences that will be characterized using a protein sequence similarity network will result in the largest catalog of substrate specificity data available to date, which in turn will enable predictions of key molecular features based on comparative sequence analysis. Third is the suggested use of directed evolution strategies such as fluorescence-activated cell sorting or antibiotic-based selection to create and evaluate million-member libraries of N-oxygenases after targeted mutagenesis. By varying amino acid identity at up to six key residues of N-oxygenases simultaneously, this project should generate N-oxygenases that exhibit superior activity on desired aromatic or heterocyclic precursors. The project team will accomplish this by designing a transcription factor that responds selectively to the desired nitro compounds at relevant concentrations. The lab has a strong track record of publications in all three of these areas, and these strategies collectively offer a transformational approach to the study of nitro compound biosynthesis.

This project will generate useful knowledge and tools for the DoD and the broader scientific community. A basic understanding of nitro compound stability in the presence of bacterial cells is lacking in the literature and has relevance spanning the discovery of new enzymes in natural products synthesis to the specificity and activity of nitroreductase enzymes that are employed to convert pro-drugs into active chemotherapeutics. In addition, this study would advance the fundamental knowledge of sequence specificity relationships in the non-heme diiron N-monooxygenase enzyme family, which is currently only available to a limited extent for a few model enzymes. Besides new knowledge, this work will create several kinds of tools useful to the DoD, industrial biotechnology, and environmental agencies, including a bacterial strain and enzymes that can be used for nitro compound biosynthesis as well as a biosensor. (Anticipated Project Completion - 2028)