Application of nitroarene dioxygenases in the design of novel strains that degrade chloronitrobenzenes.
Bottom Line: 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.
Affiliation: Department of Microbiology, College of Biological Sciences, University of California, Davis, CA 95616, USA.Show MeSH
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Mentions: Ralstonia sp. JS705 strains carrying plasmids expressing wild‐type NBDO or the NBDO‐F293Q variant were able to grow on all three CNB isomers as sole carbon sources (Table 2). Neutral resin (XAD‐7) was included to mitigate the toxicity of CNBs. 3CNB was the best substrate for both strains (Table 2, Fig. 4A), and nitrite release correlated with growth (Fig. 4B). JS705(pKSJ114) carrying NBDO‐F293Q grew 23% faster on 3CNB than the strain carrying wild‐type NBDO (Table 2), which is consistent with the activities of the two enzymes with 3CNB. In comparison, growth on 4‐chlorocatechol was inhibited even in the presence of XAD‐7, with cultures reaching a maximum cell density of 0.084 ± 0.004 with a 48 h doubling time. This is not a surprising result, as it is well known that catechols are cytotoxic even at low concentrations (Haigler et al., 1988; Munoz et al., 2007). The two strains were also capable of growth on all three CNBs as sole nitrogen sources in liquid cultures with succinate as the carbon source (data not shown).
Affiliation: Department of Microbiology, College of Biological Sciences, University of California, Davis, CA 95616, USA.