Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst

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Abstract

At present, Lpp-OmpA-mediated surface display has opened a new dimension in the development of whole-cell factories. Here, we report the surface display of methyl parathion hydrolase (MPH) and enhanced green fluorescent protein (EGFP) fusions (60 kDa) by employing the Lpp-OmpA chimera as an anchoring motif. A broad-host-range vector, pLOMG33, coding for Lpp-OmpA-MPH-GFP fusion protein was constructed for targeting the fusion protein onto the surface of Escherichia coli. The surface localization of fusion protein was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. The surface-exposed fusion protein retains the MPH activity and GFP fluorescence. Anchorage of macromolecule fusions on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli with surface-expressed MPH-GFP has two major advantages over the same strain expressing cytosolic MPH-GFP, including 7-fold higher whole-cell activity and 2-fold stronger fluorescence. Moreover, the construct pLOMG33 can potentially be applied to various bacterial species for enhancing field use. This is the first report on the presentation of GFP fusions on the cell surface by Lpp-OmpA. Our results suggest that Lpp-OmpA is a useful tool for the functional display of macromolecule passenger proteins on the cell surface.

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