Transgenic Research

, Volume 9, Issue 1, pp 43–54 | Cite as

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

  • Ziyu Dai
  • Brian S. Hooker
  • Daniel B. Anderson
  • Steven R. Thomas


The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.

Acidothermus cellulolyticus cellulase endoglucanase (E1) Nicotiana tabacum heterologous expression leaf specific promoter RbcS-3C chloroplast transit peptide RbcS-2A alfalfa mosaic virus 5′-untranslated leader 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adney WS, Thomas SR, Himmel ME, Baker JO, Chou Y-C (1998) Method for increasing thermostability in cellulase enzymes. U.S. Patent 5,712,142, Jan 29.Google Scholar
  2. An G, Ebert PR, Ha S-B (1988) Binary vectors. In: Gelvin SB and Schilperoort RA (eds). Plant Molecular Biology Mannual. (pp. 1–19) Kluwer Academic Publishers, Dordrecht, The Netherlands.Google Scholar
  3. An G (1995) Binary Ti plasmid vectors In: Gartland KMA and Davey MR (eds). Methods in Molecular Biology. Vol 44 (pp. 47–58): Agrobacterium protocols. Humana Press Inc., Totowa, NJ.Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.Google Scholar
  5. Chu C, Dai Z, Ku MSB, Edwards GE (1990) Induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum by abscisic acid. Plant Physiol 93: 1253–1260.Google Scholar
  6. Dai Z, Edwards GE, Ku MSB (1992) Control of photosynthesis and stomatal conductance in Ricinus communis L. (castor bean) by leaf to air vapor pressure deficit. Plant Physiol 99: 1426–1434.Google Scholar
  7. Dai Z, Ku MSB, Zhang D, Edwards GE (1994), Effects of growth regulators on the induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L. Planta 192: 287–294.Google Scholar
  8. Dai Z, An G (1995) Induction of nopaline synthase promoter activity by H2O2 has no direct correlation with salicylic acid. Plant Physiol 109: 1191–1197.Google Scholar
  9. Dai Z, Hooker BS, Quesenberry RD, Gao J (1999) Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli. Appl Biochem Biotech 77–79: 689–699.Google Scholar
  10. Duff SJ, Murray WD (1996) Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Bioresour Tech 55: 1–33.Google Scholar
  11. Gilbert HJ, Hazlewood GP (1993) Bacterial cellulase and xylanases. J Gen Microbiol 139: 187–194.Google Scholar
  12. Herbers K, Wilke I, and Sonnewald U (1995) A thermostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily purified. Bio/Technol 13: 63–66.Google Scholar
  13. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary vector strategy based on separation vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–181.Google Scholar
  14. Jensen LG, Olsen O, Kops O, Wolf N, Thomsen KK, Wettstein DV (1996) Transgenic barley expressing a protein-engineering, thermostable (1,3-1,4)-β-glucanase during germination. Proc Natl Acad Sci USA 93: 3487–3491.Google Scholar
  15. Lastick (deceased), Stanley M, Tucker, Melvin P, Grohmann, Karel (1996) Cloning of cellulase genes from Acidothermus cellulolyticus. US5514584, May 7.Google Scholar
  16. Liu J-H, Selinger B, Cheng K-J, Beauchemin KA, Moloney MM (1997) Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus Neocallimastix patriciarum. Mol Breed 3: 463–470.Google Scholar
  17. Lynd LR, Cushman JH, Nichols RJ, Wyman CE (1991) Fuel ethanol from cellulosic biomass. Science 251: 1318–1322.Google Scholar
  18. Mohagheghi A, Grohmann K, Himmel M, Leighton L, Updegraff D. (1986) Isolation and characterization of a new genus of thermophilic acidophilic cellulolytic bacteria, Acidothermus cellulolyticus, gne. nov., sp. nov. Int J Sys Bacteriol 36: 435–443.Google Scholar
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497.Google Scholar
  20. Pichersky E, Bernatzky R, Cashmore AR, Gruissem (1986) Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 82: 3880–3884.Google Scholar
  21. Sakon J, Adney WS, Himmel ME, Thomas SR, Karplus A (1996) Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochem 35: 10648–10660.Google Scholar
  22. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  23. Sugita M, Manzara T, Pichersky E, Cashmore A, Gruissem W (1987) Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato. Mol Gen Genet 209: 247–256.Google Scholar
  24. Thomas SR, Laymon RA, Himmel ME (1996) Gene coding for the E1 endoglucanase. US5536655, July 19.Google Scholar
  25. Tucker MP, Mohagheghi A, Grohman K, Himmel ME (1989) Ultra-thermostable cellulases from Acidothermus cellulolyticus: Comparison of temperature optima with previously reported cellulases. Bio/Technol 7: 817–820.Google Scholar
  26. Von Caemmerer SV, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–387.Google Scholar
  27. Verwoerd TC, van Paridon PA, van Ooyen AJJ, van Lent JWM, Hoekema A, Pen J (1994) Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109: 1199–1203.Google Scholar
  28. Ziegelhoffer T, Will J, Austin-Phillips S (1999) Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Mol Breed 5: 309–318.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Ziyu Dai
    • 1
  • Brian S. Hooker
    • 1
  • Daniel B. Anderson
    • 1
  • Steven R. Thomas
    • 2
  1. 1.Pacific Northwest National LaboratoryRichland
  2. 2.National Renewable Energy LaboratoryGolden

Personalised recommendations