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 that have nitroreductase activity in E. coli. Through combinatorial gene deletions of candidate nitroreductase targets, we engineered a collection of Nitro Aromatic Reductase Knockout Strains (NARKOS). With five deletions in the MG1655 progenitor strain (MG1655-NARKOS.Δ5), we observed retention of 1 mM of meta-nitrobenzoic acid and para-nitrobenzoic acid 20 h after addition to live cells compared to complete instability when added to cultures of the progenitor MG1655 strain. However, some compounds such as the di-nitro compound 3,4-dinitrobenzoic acid remained rapidly degraded in cultures of this engineered strain. Therefore, we performed additional inactivations of candidate nitroreductase genes, targeting as many as 12. With this MG1655-NARKOS.Δ12 strain, we enabled the retention of as much as 50% of the di-nitro compound supplemented at 1 mM after 20 h. We also demonstrate that we can use genome engineering to stabilize another set of compounds that are otherwise fully degraded: nitroaldehydes. We achieve more than 50% retention of multiple nitroaldehydes 4 h after supplementation at 1 mM concentration to cell cultures by performing the NARKOS gene deletions in the E. coli RARE strain background. This includes the versatile ortho-nitrobenzaldehyde synthon for photochemistry applications. Using these strains, we show that the biosynthesis of certain nitroaromatic compounds in live cells can be improved by up to 10-fold. Finally, we show that the use of the NARKOS strains can improve the dynamic range of nitro compound detection by genetically encoded biosensors. 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.

Michaela Jones

University of Delaware College of Engineering

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