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.
A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate 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 control strain 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.
Yang
Chao
C
State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Freudl
Roland
R
Qiao
Chuanling
C
Mulchandani
Ashok
A
D043303:Aryldialkylphosphatase / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology*; D001673:Biodegradation, Environmental; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000502:physiology*; D008743:Methyl Parathion / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology*; D021381:Protein Transport
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009943:Organophosphorus Compounds; C063113:ice nucleation protein; C408585:twin-arginine translocase complex, E coli; D008743:Methyl Parathion; D043303:Aryldialkylphosphatase