Biodegradation of p-nitrophenol by engineered strain

Abstract

p-Nitrophenol (PNP) is an important environmental pollutant and can causes significant environmental and health risks. Compared with the traditional methods, biodegradation is a useful one to completely remove the harmful pollutants from the environment. Here, an engineered strain was first constructed by introducing PNP biodegradation pathway via the hydroquinone (HQ) pathway into Escherichia coli. In the engineered strain BL-PNP, PNP was completely degraded to β-ketoadipate and subsequently enter the metabolites of multiple anabolic pathways. The high tolerance and rapid degradation ability to PNP enable the engineered strain to have the potential to degrade toxic substances. The engineered strain created in this study can be used as a functional strain for bioremediation of PNP and potential toxic intermediates, and the method of assembling aromatic hydrocarbons metabolic pathway can be used to eradicate nitroaromatic pollutants in the environment.

Keywords: Bioremediation; Degradation; E. coli; Multigene metabolic engineering; p-Nitrophenol.

Fig. 1

PNP degradation pathway and the two general pathways via hydroquinone or hydroxyquinol. PNP4O: p-nitrophenol 4-monooxygenase, (1.14.13.-); BQR: p-benzoquinone reductase, (1.6.5.-); H1,2O: hydroquinone 1,2-dioxygenase, (1.13.11.-); 4HSD: γ-hydroxymuconic semialdehyde dehydrogenase, (1.2.1.61); MAR: maleylacetate reductase, (1.3.1.32); N4O: 4-nitrocatechol 4-monooxygenase, (1.14.-); PNP2O: p-nitrophenol 2-monooxygenase, (1.14.13.29); QR: 2-hydroxy-1,4-benzoquinone reductase, (1.14.-); 1,2,4BTO: 1,2,4-benzenetriol dioxygenase, (1.13.11.37). The red arrows show enzyme actions designed for engineered strain, the dotted arrows show spontaneous reactions in bacteria

Fig. 2

Schematic representation of recombinant vectors used in E. coli transformation. pnpAS (p-nitrophenol 4-monooxygenase), pnpBS (p-benzoquinone reductase), pnpCS (hydroquinone 1,2-dioxygenase), pnpDS (γ-hydroxymuconic semialdehyde dehydrogenase), pnpES (maleylacetate reductase)

Fig. 3

a Expression of transgenes by PCR using the plasmid extracted from BL-control or BL-PNP as template (M, DL2000). b Expression of transgenes by quantitative real-time PCR using cDNA of BL-control or BL-PNP as template

Fig. 4

Degradation of PNP with different concentrations in the engineered strain BL-PNP, and the growth of the strain in the M9 media containing different concentration of PNP. The solid lines show the degradation of PNP, and the dotted lines show the growth of the engineered strain. Black diamond, 1 mM PNP; black square, 5 mM PNP; black up-pointing triangle, 10 mM PNP

Fig. 5

Degradation of 1 mM PNP. PNP was converted to β-ketoadipate via HQ pathway as designed. a Degradation of PNP and formation of HQ. The solid lines show the change of the metabolites in the engineered strain BL-PNP, and the dotted lines show the one in control strain. Black diamond, the degradation of PNP; black up-pointing triangle, the formation of HQ. b GC–MS analysis of β-ketoadipate concentration at different times in the engineered strain BL-PNP