Engineered E. coli strains keep fragile nitro compounds alive for green chemistry

Nitroaromatic compounds, or nitroarenes, sit at the heart of countless industrial products, from pharmaceuticals and agrochemicals to dyes and energetic materials. Yet for all their commercial importance, these molecules have long been notoriously difficult to make using biology. The reason is deceptively simple: when nitroarenes encounter living microbial cells, the cells rapidly strip away the very nitro groups that make the compounds valuable, reducing them through nitroso and hydroxylamine intermediates all the way down to amines. Now, a team of researchers at the University of Delaware has reported a systematic solution, engineering strains of Escherichia coli that can retain, and even biosynthesize, a remarkably diverse set of nitroaromatic chemicals. The work, published in Nature Chemistry, could reshape how chemists think about building nitro groups under mild, aqueous, enzyme-friendly conditions.

The conventional route to nitroarenes relies on nitric acid, often paired with sulfuric acid, in aggressive industrial nitration reactions. These processes demand stringent heat management, generate environmental hazards, and struggle with regioselectivity and functional group tolerance. Biology, in principle, offers a greener alternative: enzymes that install nitro groups in water at ambient temperatures, with exquisite selectivity, potentially using molecular oxygen as the only co-reactant and water as the byproduct. But two obstacles have stalled progress. First, only around 200 nitro-containing natural products are known, limiting the pool of enzymes available for discovery, although specialized N-oxygenases such as CmlI, AurF, ObiL and PrnD, along with cytochrome P450 enzymes like TxtE, have begun to fill the toolkit. Second, and more fundamentally, common microbial hosts like E. coli erase nitro functionality almost as fast as it can be made.

That erasure is the work of nitroreductases, or NTRs, a sprawling and redundant family of flavin-dependent enzymes embedded in the broader redox physiology of the cell. Many of these enzymes also handle quinone reduction, redox mediation and oxidative-stress defence, which makes them hard to identify and harder to eliminate. Previous studies had shown that deleting multiple NTR genes could stabilize certain prodrugs or photocaged amino acids, but no one had quantitatively mapped how nitroarene stability scales with the number of deletions and incubation time. The Delaware team, led by Aditya Kunjapur, set out to close that gap with a combinatorial genome engineering campaign they dubbed the NARKOS collection, short for nitroaromatic reductase knockout strains.

Using multiplexed automatable genome engineering, or MAGE, the researchers introduced translational knockouts into wild-type E. coli MG1655 (DE3). Their first strain, MG1655-NARKOS.Δ5, inactivated five previously documented NTR genes: nfsA, nfsB, azoR, nemA and ydjA. They then supplemented cultures of both wild-type and engineered cells with one millimolar concentrations of 20 simple nitroarenes, varying the position and number of nitro groups and the identity of other ring substituents, and measured compound retention by HPLC after 20 hours of incubation at 37 degrees Celsius. The results were striking. For several compounds, including many para-substituted nitroarenes, the five-deletion strain retained more than 90 percent of the starting material, compared with partial or no retention in wild-type cultures. Meta-nitrobenzoic acid was retained only in the engineered strain, while ortho-substituted isomers often showed comparable stability in both backgrounds, revealing that nitro group positioning strongly modulates susceptibility to cellular reduction.

The di-nitrobenzoic acids in the panel proved more stubborn, exposing the limits of the first five deletions. Compound 18 and 19 showed 7.4-fold and 3.3-fold stability improvements in the engineered strain, but complete retention remained out of reach, and 3,4-dinitrobenzoic acid, compound 20, was completely unstable in both strains despite being stable in cell-free medium. This pointed to additional, less-characterized endogenous enzymes with high activity on dinitro substrates. Intriguingly, the nitro-substituted amino acids in the panel were largely stable even in wild-type cells, suggesting that the zwitterionic amino acid scaffold can shield the nitroaromatic motif from enzymatic recognition. The team also found a strong correlation, with an R-squared of 0.91, between cellular instability and single-electron reduction potential for the six compounds with measured values, supporting a model in which the endogenous flavoprotein pool acts as a broad-spectrum electron-acceptor filter governed primarily by redox thermodynamics.

To push further, the researchers stacked additional knockouts onto the Δ5 background, constructing strains with nine, eleven and twelve deleted genes targeting quinone reductases and other oxidoreductases implicated in collateral nitro reduction. The gains were substantial. Compound 10 reached 93 percent retention in the Δ9 through Δ12 strains, roughly double its stability in Δ5. Compound 18 hit 98 percent retention in Δ9, and compound 20 improved 28-fold in Δ12 relative to Δ5. Complementation experiments, in which individual deleted genes were re-expressed on plasmids, confirmed that enzymes such as NfsA, NfsB, NemA, MdaB, YieF and KefF each abolish retention when present. A further screen of 13 candidate genes identified SsuE and Fpr as additional contributors, although deleting them alongside kefG in a Δ15 strain did not improve stability beyond Δ12, hinting that the remaining reduction capacity had been largely exhausted. Resting whole cells of the engineered strains, which carry less reductive power than actively growing cultures, fully retained compound 19 and retained 80 percent of compound 20 after 20 hours.

Nitroaromatic aldehydes posed a second, distinct challenge, because E. coli rapidly reduces aldehydes through aldo-keto reductases and alcohol dehydrogenases. The team therefore combined their NTR knockouts with the RARE strain, a previously engineered aldehyde-stabilizing chassis lacking six such reductase genes. The resulting RARE-NARKOS strains carry up to 15 total gene deletions. When supplemented with ortho-, meta- and para-nitrobenzaldehyde, wild-type cells, the Δ5 strain and even the original RARE strain retained less than 5 percent of each compound after just four hours. RARE-NARKOS.Δ5, by contrast, retained 52, 78 and 13 percent of the three isomers respectively, and RARE-NARKOS.Δ9 pushed ortho-nitrobenzaldehyde retention to 74 percent. Additional engineering revealed substrate-specific effects: extra NTR deletions most benefited the ortho isomer, while extra aldehyde-reductase deletions most benefited the para isomer, a 6.5-fold improvement after 20 hours.

A critical question for any industrial platform is whether such heavy genome editing compromises fitness. The answer, encouragingly, was mostly no. In rich complex and defined media, all engineered strains grew at rates comparable to their progenitors, and heterologous protein expression, measured with a fluorescent reporter, was unaffected. Only in minimal M9 medium did the deeper knockout strains show reduced growth rates, suggesting a cumulative metabolic burden that would need optimization for nutrient-limited processes. Within the rich media typically used for biosynthesis, however, the NARKOS chassis performed robustly, clearing the way for its real purpose: enabling nitroarene chemistry inside living cells.

The applications demonstrated were among the most compelling aspects of the study. When the researchers expressed the N-oxygenase AurF in wild-type E. coli to oxidize para-aminobenzoic acid, nitro product titres were barely detectable. In MG1655-NARKOS.Δ5, titres rose 17-fold, and in Δ12 they rose 41-fold. Screening 21 N-oxygenase homologues in the Δ12 background yielded detectable nitro production for two target compounds under 10 and 13 conditions respectively, whereas the identical screen in wild-type cells produced nothing detectable at all. The team also converted nitrobenzoic acids into nitrobenzaldehydes using a carboxylic acid reductase, achieving production and accumulation in every RARE-NARKOS strain while the progenitor RARE strain produced nothing. Finally, they coupled biosynthesis with genetic code expansion, using a transaldolase, a phenylserine dehydratase and an aminotransferase to convert nitrobenzaldehyde precursors into nitrophenylalanines, which were then site-specifically incorporated into a GFP reporter via orthogonal translation systems. Titres of ortho-, meta- and para-nitro-phenylalanine rose 5.2-fold, 38.7-fold and 3.2-fold in the engineered strain, with fluorescent outputs increasing 6-fold, 5-fold and 30-fold over no-substrate controls.

The broader implications extend across biocatalysis, metabolic engineering and synthetic biology. Nitro-substituted non-standard amino acids include light-responsive building blocks that can cleave polypeptide chains on irradiation, and motifs that enable immunochemical termination of protein self-tolerance, making scalable access to these molecules commercially meaningful. The semi-synthesis route demonstrated here offers a potential cost advantage for nitrophenylalanines that currently resemble specialty rather than commodity chemicals, and provides an entry point to non-commercial analogues. More fundamentally, the work establishes a design principle: rather than hunting for a single master nitroreductase, engineers can eliminate redundant classes of endogenous reductases in layers, tailoring the chassis to the redox lability of the target molecule. The same logic, the authors note, may extend to other redox-labile, electron-accepting functional groups beyond nitroarenes. As enzymatic nitration discovery accelerates on platforms like NARKOS, the long-standing divide between harsh industrial nitration chemistry and gentle biological synthesis may finally begin to close.

Subject of Research: Engineering nitroreductase-deficient Escherichia coli for retention and biosynthesis of nitroaromatic compounds

Article Title: Retention and biosynthesis of diverse nitroarenes in Escherichia coli after combinatorial nitroreductase gene deletions

Article References: Anderson, S. R., Gupta, S. P., Butler, N. D., Jones, M. A., & Kunjapur, A. M. (2026). Retention and biosynthesis of diverse nitroarenes in Escherichia coli after combinatorial nitroreductase gene deletions. Nature Chemistry. https://doi.org/10.1038/s41557-026-02266-8

Image Credits: AI Generated

DOI: 10.1038/s41557-026-02266-8

Keywords: nitroarenes, nitroreductases, Escherichia coli, genome engineering, biocatalysis, metabolic engineering, synthetic biology, N-oxygenases, genetic code expansion, non-standard amino acids, green chemistry, flavoproteins