Construction of a Green Fluorescent Protein (GFP)-Marked Multifunctional Pesticide-Degrading Bacterium for Simultaneous Degradation of Organophosphates and γ-Hexachlorocyclohexane

Abstract

An autofluorescent whole-cell biocatalyst capable of simultaneously degrading organophosphates (OPs) and γ-hexachlorocyclohexane (γ-HCH) was constructed by display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion on the cell surface of a γ-HCH-degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-OPH-GFP fusion was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibits cell growth nor affects cell viability. In sterile and nonsterile soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days when inoculated with the engineered UT26, and the strain could be easily monitored by fluorescence during bioremediation. These results indicate that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of environments contaminated with multiple pesticides.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli

“…Proteinases cannot cross the outer membrane of the cell, and, therefore, only surface-displayed proteins can be degraded by proteinases [30]. Therefore, evidence for the localization of target proteins on the cell surface can be proved by a proteinase accessibility assay.…”

Degradation strategies of pesticide residue: From chemicals to synthetic biology

“…With the surface display systems, we can expand the function of some pesticide-degrading microorganisms to cope with multiple pesticide residues. As chlorpyrifos and other organophosphorus pesticides are usually more difficult to pass through the membrane, the common method is displaying an enzyme that can degrade organophosphorus on the surface of γ-HCH degrading strains like Sphingobium japonicum UT26 [ [94] , [95] , [96] ] while constructing a multi-function bacteria. With the application of fusion protein, engineered bacteria can expand other functions to improve their practicability.…”

“…The rapid development of synthetic biology provides a powerful tool for pesticide degradation because of its advantage in diverse combination, precise control mode, and variable design strategy [ 17 ]. More and more scientists have devoted themselves to employing synthetic biology to degrade pesticide residues [ [18] , [19] , [20] , [21] , [22] ]. These works provide not only a variety of technical support for the degradation of pesticide residues beyond the limitations of original technology but also the insurance for environment and food safety.…”

Engineering Pseudomonas putida KT2440 for simultaneous degradation of carbofuran and chlorpyrifos

“…In addition, a γ‐hexachlorocyclohexane (γ‐HCH)‐degrading Sphingobium japonicum UT26 was endowed with the capacity to simultaneously degrade γ‐HCH and organophosphates by transforming with a plasmid encoding an organophosphorus hydrolase. However, plasmid instability reduces the efficacy of the recombinant stain for bioremediation of contaminated soil (Yang et al ., ).…”