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Specific Adhesion and Hydrolysis of Cellulose by Intact Escherichia coli Expressing Surface Anchored Cellulase or Cellulose Binding Domains

Abstract

The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed hi Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added hi the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.

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References

  1. 1

    Cowan, S.W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R.A., Jansonius, J.N. and Rosenbusch, J.P. 1992. Crystal structures explain functional properties of two E. coli porins. Nature 358: 727–733.

    CAS  Article  Google Scholar 

  2. 2

    Nikaido, H. 1991. Porins and specific channels of bacterial outer membranes. Mol. Microbiol. 6: 435–442.

    Article  Google Scholar 

  3. 3

    Valkonen, K.H., Vejola, J., Dagberg, B. and Uhlin, B.E. 1991. Binding of basement-membrane laminin by Escherichia coli. Mol. Microbiol. 5: 2133–2141.

    CAS  Article  Google Scholar 

  4. 4

    Olsen, A., Jonsson, A. and Normark, S. 1989. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature 338: 652–655.

    CAS  Article  Google Scholar 

  5. 5

    Brown, S. 1992. Engineered iron oxide-adhesion mutants of the Escherichia coli phage λ receptor. Proc. Natl. Acad. Sci. U.S.A. 89: 8651–8655.

    CAS  Article  Google Scholar 

  6. 6

    Newton, S.M.C., Jacob, C.O. and Stocker, B.A.D. 1989. Immune response to cholera toxin epitope inserted in Salmonella flagellin. Science 244: 70–72.

    CAS  Article  Google Scholar 

  7. 7

    Charbit, A., Sobczak, E., Michel, M.L., Molla, A., Tiollais, P. and Hofnung, M. 1987. Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria. J. Immunol. 139: 1658–1664.

    CAS  PubMed  Google Scholar 

  8. 8

    Georgiou, G., Poetschke, H.L., Stathopoulos, C. and Francisco, J.A. 1993. Engineering of microbial surfaces for biotechnology applications. Trends in Biotechnol. 11: 6–10.

    CAS  Article  Google Scholar 

  9. 9

    Clackson, T., Hoogenboom, H.R., Griffiths, A.D. and Winter, G. 1991. Making antibody fragments using phage display libraries. Nature 352: 624–628.

    CAS  Article  Google Scholar 

  10. 10

    Chiswell, D.J. and McCafferty, J. 1992. Phage antibodies: Will new “coliclonal” antibodies replace monoclonal antibodies? Trends in Biotechnol. 10: 80–84.

    CAS  Article  Google Scholar 

  11. 11

    Klauser, T., Johannes, P. and Meyer, T.F. 1992. Selective extracellular release of cholera toxin B subunit by Escherichia coli: Dissection of Neisseria Igaβ-mediated outer membrane transport. EMBO J. 11: 2327–2335.

    CAS  Article  Google Scholar 

  12. 12

    Francisco, J.A., Earhart, C.F. and Georgiou, G. 1992. Transport and anchoring of β-lactamase to the external surface of Escherichia coli. Proc. Natl. Acad. Sci. USA 89: 2713–2717.

    CAS  Article  Google Scholar 

  13. 13

    Gilkes, N.R., Kilburn, D.G., Langsford, M.L., Miller, R.C. Jr., Wakarchuk, W.W., Warren, R.A.J., Whittle, D.J. and Wong, W.K.R. 1984. Isolation and characterization of Escherichia coli clones expessing cellulase genes from Cellulomonas fimi. J. Gen. Microbiol. 130: 1377–1384.

    CAS  Google Scholar 

  14. 14

    Ong, E., Gilkes, N.R., Miller Jr., R.C., Warren, R.A.J. and Kilburn, D.G. 1992. The cellulose-binding domain (CBDCex) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide. Biotechnol. Bioeng. Submitted.

  15. 15

    Ong, E., Gilkes, N.R., Warren, R.A.J., Miller, R.C. Jr. and Kilburn, D.G. 1989. Enzyme immobilization using the cellulose-binding domain of Cellumonas fimi exoglucanase. Bio/Technology 7: 604–607.

    CAS  Google Scholar 

  16. 16

    Gilkes, N.R., Warren, R.A.J., Miller Jr., R.C. and Kilburn, D.G. 1988. Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J. Biol. Chem. 263: 10401–10407.

    CAS  Google Scholar 

  17. 17

    Zhang, Y. and Broome-Smith, J.K. 1990. Correct insertion of a simple eucaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli. Gene 96: 51–57.

    CAS  Article  Google Scholar 

  18. 18

    O'Neill, G.P., Kilburn, D.G., Warren, R.A.J. and Miller Jr., R.C. 1986. Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli. Appl. Environ. Microbiol. 52: 737–743.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Muthuria, M.L. and Hancock, R.E.W. 1985. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 31: 381–386.

    Article  Google Scholar 

  20. 20

    Stattford, M. and Bond, C.J. 1992. Selective separation of microorganisms by lectins: Yeast and concanavalin A as model system. Biotechnol. Bioeng. 40: 835–843.

    Article  Google Scholar 

  21. 21

    Ogden, K.L., Davis, R.H. and Taylor, A.L. 1992. An adjustable expression system for controlling growth rate, plasmid maintenance and culture dynamics. Biotechnol. Bioeng. 40: 1027–1038.

    CAS  Article  Google Scholar 

  22. 22

    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.) 1989. Current Protocols in Molecular Biology. John Wiley and Sons, New York.

  23. 23

    Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Masui, Y. and Inouye, M. 1984. Secretion cloning vectors in Escherichia coli. EMBO J. 3: 2437–2442.

    CAS  Article  Google Scholar 

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Francisco, J., Stathopoulos, C., Warren, R. et al. Specific Adhesion and Hydrolysis of Cellulose by Intact Escherichia coli Expressing Surface Anchored Cellulase or Cellulose Binding Domains. Nat Biotechnol 11, 491–495 (1993). https://doi.org/10.1038/nbt0493-491

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