Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil
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Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (106 cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.
KeywordsOrganophosphate Pyrethroid Pseudomonas putida KT2440 Bioremediation
The authors gratefully acknowledge the financial support from National Key Basic Research Program of China (No. 2012CB725204), National High Technology Research and Development Program of China (No. 2013AA06A210), National Natural Science Foundation of China (Nos. 31300032, 31470213 and 31170030), Project of Tianjin, China (Nos. 13JCQNJC09700, 13JCZDJC27800 and 20140609), Open Fund of State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University (No. MMLKF13-06), and Open Fund of State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences (No. SKLMR-20130604).
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