Synthetic organophosphates (OPs) are widely used to control agricultural pests and account for ~38% of the total pesticides used globally. In the United States alone, over 40 million kilograms of OP pesticides are consumed annually. OPs are acute neurotoxins by virtue of their potent inhibition of acetylcholinesterase (AChE). OP pesticides are mostly liposoluble and pose a hazard to humans through accumulation in the food chain. In a recent study carried out by Chuanling Qiao’s research group, E. coli strain XL1-Blue was genetically engineered to co-express methyl parathion hydrolase (MPH) and organophosphorus hydrolase (OPH) using two compatible vectors. Since MPH and OPH have different substrate specificities, the engineered strain acquires an enlarged substrate range for OPs. The cell membrane acting as a permeability barrier limits interaction between the pesticides and OP hydrolase residing within the cell. To alleviate the substrate uptake limitation, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. For this purpose, MPH was fused to the twin-arginine signal peptide of trimethylamine N-oxide reductase (TorA) and OPH to the N- and C-terminal domains of InaV protein (INPNC). The correct localization of MPH and OPH was demonstrated by cell fractionation, Western blot, and OP hydrolase assay. A mixture of diethyl and dimethyl OPs (0.2 mM each) could be degraded completely by the engineered strain within 5 h. The engineered strain capable of targeting MPH and OPH to extracytosolic compartment possesses a broader substrate range than strains expressing either one of the hydrolases and overcomes the mass transport limitation of OP pesticides across the cell membrane. In conclusion, the engineered strain has enormous potential for use as a whole-cell biocatalyst for detoxification of a mixture of OP pesticides. This work has been published in Applied and Environmental Microbiology, 2010, 76, 434-440.

This work was financially supported by the 863 Hi-Tech Research and Development Program of the People’s Republic of China (Nos. 2007AA06Z335 and 2007AA061101).