Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion

Abstract

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.

Surface display of multi-epitopes synthetic gene of Mycobacterium tuberculosis on Salmonella typhi Ty21a using a pgsA anchor protein

“…The PgsA carrier protein is a poly-γ-glutamic acid (PGA) derived from Bacillus subtilis chungkookjang, whereas the HCE promoter is a highly constitutive expression promoter derived from Geobacillus toebi (Poo et al, 2002;Narita et al, 2006). This HCE-PgsA surface display expression system has been shown useful in the area of protein expression as well as vaccine development.…”

“…This HCE-PgsA surface display expression system has been shown useful in the area of protein expression as well as vaccine development. Narita et al (2006) has shown that, HCE-PgsA system is able to display a 78 kDa protein on E. coli cells in the active form. The author also claims that PgsA protein is the first carrier protein that is able to display such a huge enzyme in conformationally active form on the cell surface of E. coli.…”

“…A number of carrier proteins such as ice nucleation protein (Inp) (Kim et al, 2001;Lee et al, 2000;Kwak et al, 1999), outer membrane protein (Omp) (Haddad et al, 1995) and autotransporter (Rutherford and Mourez, 2006;Kramer et al, 2003) have been used to display foreign protein on bacterial cell surfaces. The use of different carrier proteins frequently cause different physiological effects on host cells including growth defects and destabilization of the outer membrane (Lee et al, 2003;Narita et al, 2006).…”

“…The efforts to develop better carrier proteins are still in progress and new carrier proteins have been developed recently including PrsA (Kim et al, 2005) and PgsA (Ashiuchi et al, 2004;Narita et al, 2006). PgsA protein is a carrier protein derived from poly-γ-glutamate (PGA) synthetase complex of Bacillus subtillis (Ashiuchi et al, 2004).…”

“…PgsA protein is a carrier protein derived from poly-γ-glutamate (PGA) synthetase complex of Bacillus subtillis (Ashiuchi et al, 2004). Narita et al have shown that, PgsA protein is able to display proteins as large as -77 kDa in active form on the surface of Escherichia coli (Narita et al, 2006).…”