(605e) The Elimination of Nitroreductases in E. coli enables the Retention of Many Nitro Compounds for Biosynthesis or Biosensing Applications

Nitro groups are important components of small molecule pharmaceuticals, energetic materials, commodity chemicals, and natural products such as antibiotics. One challenge associated with the detection or production of nitro compounds using microbial hosts is their context-dependent instability in and around cells. In this study, we measured the stability of diverse nitroaromatic compounds in the presence of E. coli and found that we could improve stability substantially by engineering combinatorial gene inactivations. The identity and position of the other ring substituents strongly affects stability, which reflects the preferences of natively expressed enzymes with nitroreductase activity in E. coli. For example, we observed retention of 1 mM of meta-nitrobenzoic and para-nitrobenzoic 20 h after addition to a strain that contained five inactivated genes, compared to complete instability when added to cultures of the parent MG1655 strain. Furthermore, in an engineered strain with eleven gene inactivations, we enable the detection of 3,4-dinitrobenzoic acid, a di-nitro compound which was otherwise unstable in both the parent strain and the strain with only five gene inactivations 20 h after its addition. We refer to this engineered Nitro Aromatic Reductase Knockout Strain as MG1655-NARKOS. In addition to enabling the retention of some nitrobenzoic acids in cell culture, here we demonstrate that we can enable the detection of supplemented nitroaldehydes 4 h after addition to cells. More than 50% of the 1 mM concentration initially supplied to cells for multiple nitroaldehydes is observed, including the versatile ortho-nitrobenzaldehyde synthon for photochemistry applications, by performing gene inactivations in a strain that contained aldehyde reductase inactivations, denoted RARE-NARKOS. Using these strains, we show for the first time that the biosynthesis of certain nitroaromatic compounds in live cells can be improved by up to 10-fold. Finally, we improved the dynamic range of nitroaldehyde detection by genetically encoded biosensors expressed in cells using our RARE-NARKOS host. Our study represents a major advance in the understanding of nitro compound metabolism and the ability of engineered biological systems to access this important chemistry.