The objective of this project is to populate the synthetic biology toolbox with well characterized genes for the biosynthesis of nitroorganic compounds. This project will accomplish this by first identifying candidate fungi that produce nitroorganic compounds then further characterizing the fungi and their nitroorganic product profile. Comparative transcriptomics studies of the fungi will be performed to identify the biosynthetic genes. This will be followed by cloning and expression of candidate genes to demonstrate their biosynthetic utility and to allow characterization of the nitroorganic product profile of the enzymes.

This project will identify genes and enzymes involved in the biosynthesis of nitroorganic compounds in fungi using transcriptomics and genomics followed by expression and analysis of those genes. Research will begin with the relatively well characterized 3-nitropropanoic acid producing fungus Aspergillus oryzae to demonstrate the effectiveness of the approach. The project will subsequently identify other candidate nitroorganic producing fungi and isolate the genes involved in nitroorganic biosynthesis by feeding them into the gene discovery and parts characterization pipeline. In addition, this project seeks to identify other genes important in regulation of production and export of nitroorganic compounds in fungi. These tasks will build the synthetic biology toolbox for synthesis of various natural or xenobiotic nitroorganics in A. niger or other suitable platform organisms.

This foundational research will begin to fill the synthetic biology toolbox of "relevant and new biocatalysts" (i.e., enzymes and the genes that encode them) to meet the needs of the Department of Defense in the production of nitroorganics. The resulting bioprocesses would exhibit high selectivity without the use of solvents, metal catalysts, or strong acids and utilize sugars as carbon sources instead of petroleum. (Anticipated Project Completion - 2017)