Widespread application of chloronitrobenzenes as feedstocks for the production of industrial chemicals and pharmaceuticals has resulted in extensive environmental contamination with these toxic compounds, where they pose significant risks to the health of humans and wildlife. While biotreatment in general is an attractive solution for remediation, its effectiveness is limited with chloronitrobenzenes due to the small number of strains that can effectively mineralize these compounds and their ability to degrade only select isomers. To address this need, we created engineered strains with a novel degradation pathway that reduces the total number of steps required to convert chloronitrobenzenes into compounds of central metabolism. We examined the ability of 2-nitrotoluene 2,3-dioxygenase from Acidovorax sp. strain JS42, nitrobenzene 1,2-dioxygenase (NBDO) from Comamonas sp. strain JS765, as well as active-site mutants of NBDO to generate chlorocatechols from chloronitrobenzenes, and identified the most efficient enzymes. Introduction of the wild-type NBDO and the F293Q variant into Ralstonia sp. strain JS705, a strain carrying the modified ortho pathway for chlorocatechol metabolism, resulted in bacterial strains that were able to sustainably grow on all three chloronitrobenzene isomers without addition of co-substrates or co-inducers. These first-generation engineered strains demonstrate the utility of nitroarene dioxygenases in expanding the metabolic capabilities of bacteria and provide new options for improved biotreatment of chloronitrobenzene-contaminated sites.

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

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