Development of a Whole-Cell Biocatalyst/Biosensor by Display of Multiple Heterologous Proteins on the Escherichia coli Cell Surface for the Detoxification and Detection of Organophosphates

Abstract

This paper reports the codisplay of organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH)-green fluorescent protein (GFP) fusion on the cell surface of Escherichia coli using the truncated ice nucleation protein (INPNC) and Lpp-OmpA as the anchoring motifs. The surface localization of both OPH and MPH-GFP was demonstrated by cell fractionation, Western blot analysis, protease accessibility experiment, and immunofluorescence microscopy. Anchorage of the foreign proteins on the outer membrane neither inhibits cell growth nor affects cell viability. The recombinant strain can be used as a whole-cell biocatalyst and showed a broader substrate range than strains expressing either OPH or MPH. A mixture of six organophosphorus pesticides (OPs) (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes the recombinant strain a promising candidate for detoxification of OPs. The fluorescence of surface-displayed GFP is very sensitive to environmental pH change. Because hydrolysis of OPs by OPH or MPH generates protons, the recombinant E. coli could be used as a whole-cell biosensor for the rapid detection of OPs by evaluating fluorescence changes as a function of OP concentrations.

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

“…Secretomes are traditionally studied in vitro , using biochemical and proteomics approaches, and in silico , using bioinformatic tools. Surface display screening methods and reporter fusion systems ( Georgiou et al, 1997 ; Chen and Georgiou, 2002 ; Åvall-Jääskeläinen et al, 2003 ; Lee et al, 2003 ; Wernérus and Ståhl, 2004 ; Liu et al, 2013 ), as well as phage-display based systems ( Rosander et al, 2002 , 2011 ; Jacobsson et al, 2003 ; Bjerketorp et al, 2004 ), described in detail in section 3, have also been used for screening, identifying, and characterizing secretome proteins.…”

Engineering Novel and Improved Biocatalysts by Cell Surface Display

“…11,75,118 A number of successful whole-cell biocatalysts using direct display of OP-degrading enzymes have been developed, 75,119,120 including a system utilizing co-display of two enzymes for simultaneous OP detection and degradation. 118 In this system, organophosphorus hydrolase (OPH) and a methyl parathion hydrolase (MPH)–GFP fusion were co-displayed using truncated INP and Lpp-OmpA anchors, respectively. Because GFP fluorescence intensity is sensitive to environmental pH and the degradation of OPs by OPH and MPH generates protons, the surface-engineered E. coli strain was able to detect and degrade a broad range of OPs by monitoring changes in GFP fluorescence.…”

Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1

“…Among them, surface display of specific proteins on microbes for the biosorption of heavy metal ions has emerged as a new research hotspot (Wu et al, 2008;Saleem et al, 2008;Biondo et al, 2012). Further, the surface display technique can be employed to establish simple biosensors for detection of contaminants including heavy metals (Lee et al, 2003;Liu et al, 2013;Wei et al, 2014). The earlier study shows that mercury-resistant strain Pseudomonas aeruginosa owns excellent mercury adsorption capacity (Chang and Hong, 1994).…”