Applied and Environmental Microbiology, January 2010, p. 434-440, Vol. 76, No. 2

Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

State Key Laboratory of Integrated Management of Pest Insects Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China, Institut für Biotechnologie, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany, Department of Chemical and Environmental Engineering, University of California, Riverside, California

Received 7 September 2009/ Accepted 10 November 2009

A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase(MPH) was constructed for the first time by cotransforming twocompatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broadersubstrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, 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. The resulting recombinant strain showed sixfold-higher whole-cell activity than the controlstrain expressing cytosolic OP hydrolases.The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays.

No growth inhibition was observed for the recombinant E.coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E.coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.