Production of N-Nitroglycine by Streptomyces noursei JCM 4701

Streptomyces noursei JCM 4701 utilizes bionitration to produce N-nitroglycine, a rare nitramine natural product structurally similar to the energetic materials nitroguanidine and dinitrourea. Identification of the enzymes that produce the nitro groups of these energetic materials would provide a green chemistry alternative for reducing the environmental impact relative to currently employed chemical nitration reactions. However, nothing is presently known about the biosynthetic pathway for this compound. Using S. noursei as a host, we have demonstrated N-nitroglycine synthesis and verified production using HPLC and HR-MS. Growth conditions have been optimized for maximal N-nitroglycine production, and we have shown that this secondary metabolite is produced near the end of exponential growth. Labelled feeding experiments have established that glycine is the backbone precursor to N-nitroglycine, and additional feeding experiments are underway to determine the origin of the nitro group and to elucidate the full biosynthetic pathway. Initial tracer studies indicated that glutamine and nitrate are not immediate precursors. To identify the proteins involved in N-nitroglycine production, a 10.3 Mbp S. noursei draft genome containing 9156 coding sequences was assembled and the protein production dynamics of cells grown in rich media were observed across all growth phases and characterized in combination with the draft genome. This analysis indicated that 779 of the 4389 proteins identified increased in abundance in late growth-phase cells that actively produced N-nitroglycine. In light of these data, an intergeneric conjugal transfer system has been established to facilitate molecular genetic studies to confirm candidate genes responsible for the nitration reaction.