Biotechnology Letters

, Volume 34, Issue 1, pp 91–96 | Cite as

Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli

  • Diya M. Abdeljabbar
  • Hank J. Song
  • A. James Link
Original Research Paper

Abstract

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

Keywords

Fungal cellulase Fusion protein Heterologous protein expression Membrane localization 

References

  1. Arnold FH (1998) Design by directed evolution. Accounts Chem Res 31:125–131CrossRefGoogle Scholar
  2. Bayer EA, Chanzy H, Lamed R, Shoham Y (1998) Cellulose, cellulases, and cellulosomes. Curr Opin Struc Biol 8:548–557CrossRefGoogle Scholar
  3. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA 96:13703–13708PubMedCrossRefGoogle Scholar
  4. Bhikhabhai R, Pettersson G (1984) The disulfide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei. Biochem J 222:729–736PubMedGoogle Scholar
  5. Daugherty PS (2007) Protein engineering with bacterial display. Curr Opin Struc Biol 17:474–480CrossRefGoogle Scholar
  6. Denman S, Xue GP, Patel B (1996) Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II. Appl Environ Microb 62:1889–1896Google Scholar
  7. Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biot 63:258–266CrossRefGoogle Scholar
  8. Divne C, Stahlberg J, Teeri TT, Jones TA (1998) High resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol 275:309–325PubMedCrossRefGoogle Scholar
  9. Goyal A, Ghosh B, Eveleigh D (1991) Characteristics of fungal cellulases. Bioresource Technol 36:37–50CrossRefGoogle Scholar
  10. Guilvout I, Chami M, Engel A, Pugsley AP, Bayan N (2006) Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin. EMBO J 25:5241–5249PubMedCrossRefGoogle Scholar
  11. Himmel ME, Ding S, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807PubMedCrossRefGoogle Scholar
  12. Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674PubMedCrossRefGoogle Scholar
  13. Nieves RA, Ehrman CI, Adney WS, Elander RT, Himmel ME (1998) Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. World J Microb Biot 14:301–304CrossRefGoogle Scholar
  14. Osborn MJ, Munson R (1974) Separation of the inner (cytoplasmic) and outer membranes of gram-negative bacteria. Methods Enzymol 31:642–653PubMedCrossRefGoogle Scholar
  15. Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S (2005) NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307:1317–1321PubMedCrossRefGoogle Scholar
  16. Sklar JG, Wu T, Gronenberg LS, Malinverni JC, Kahne D, Silhavy TJ (2007) Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli. Proc Natl Acad Sci U S A 104:6400–6405PubMedCrossRefGoogle Scholar
  17. Xu Z, Lee SY (1999) Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif. Appl Environ Microb 65:5142–5147Google Scholar
  18. Zhang YHP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24:452–481CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Diya M. Abdeljabbar
    • 1
  • Hank J. Song
    • 1
  • A. James Link
    • 1
    • 2
    • 3
  1. 1.Department of Chemical & Biological EngineeringPrinceton UniversityPrincetonUSA
  2. 2.Department of Molecular BiologyPrinceton UniversityPrincetonUSA
  3. 3.A207 Engineering QuadranglePrinceton UniversityPrincetonUSA

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