Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

Abstract

We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.

Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

“…Moreover, a stable and regenerable cell platform is apparently conducive to retain the activity of surface-displayed enzymes [25,26]. Bacterial display systems are normally grouped into those that allow N-terminal, C-terminal, and “sandwich” fusions.…”

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

“…To prepare outer membrane proteins (Lee et al 2005), 1 ml culture broth was centrifuged at 5,000 9 g for 5 min at 4°C, the cell pellet was washed with 1 ml 10 mM sodium phosphate buffer (pH 7.2), re-centrifuged and the final cell pellet was resuspended in 0.5 ml 10 mM sodium phosphate buffer (pH 7.2). Crude extracts of recombinant E. coli cells were prepared by three cycles of sonication, followed by centrifugation at 12,000 9 g for 2 min at room temperature and the supernatant was centrifuged at 12,000 9 g for 30 min at 4°C.…”

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

“…Besides the above anchoring motifs, many others have been developed including OprF, OmpX and Lpp-OmpA [17,18,19]. As cell surface display technology can be applied to increase the extracellular β-fructofuranosidase activity, sucrose can be hydrolyzed quickly outside the cells.…”