Production of N-nitroglycine by Streptomyces noursei

Streptomyces noursei produces N-nitroglycine, a rare nitramine natural product synthesized using bionitration. N-nitroglycine is structurally similar to the energetic materials nitroguanidine and dinitrourea. Identifying new enzymes to produce the nitro groups of energetic materials would be using green chemistry to reduce the environmental impact from the currently employed chemical nitration reactions. However, the substrate and reaction pathway for N-nitroglycine were unknown. We optimized the growth conditions for maximal N-nitroglycine production and determined that this nitro compound is not produced until a late growth-phase. We developed HPLC and HR-MS to detect this compound using a chemically synthesized standard. We determined that N-nitroglyicne could be produced when the cells were grown solely in yeast extract.  Additional experiments were initiated to determine which component of yeast extract was involved in the reaction. N-nitroglycine was produced in the presence of 4-aminobenzoic acid.  Feeding experiments using labeled compounds will be used to determine whether this compound is a precursor to N-nitroglycine. S. noursei was sequenced and the draft genome is 10.3 Mbp, with 9156 coding DNA sequences. In order to identify proteins involved in N-nitroglycine production, cells were grown in rich media, and samples were removed at different times for differential proteomic characterization in combination with the draft genome. Preliminary proteomic analysis indicated that of the 4389 proteins identified, 779 were found to have increased abundance in late growth-phase cells that produced N-nitroglycine. Bioinformatics, biotransformations, and inhibitor studies are being used to identify protein candidates responsible for this nitration reaction.