Transgenic Research

, Volume 14, Issue 5, pp 627–643 | Cite as

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

  • Ziyu DaiEmail author
  • Brian S. Hooker
  • Ryan D. Quesenberry
  • Steven R. Thomas


An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5′-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5′-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.


Acidothermus cellulolyticus cellulases E1 endoglucanase post-transcriptional modification sub-cellular compartmentation transgenic tobacco 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. An, G, Ebert, PR, Ha, S-B 1988Binary vectorsGelvin, SBSchilperooort, RA eds. Plant Molecular Biology MannualKluwer Academic PublishersDordrecht, The Netherland119Google Scholar
  2. Bailey, MR, Woodard, SL, Callaway, E, Beifuss, K, Magallanes-Lundback, M, Lane, JR, Horn, ME, Mallubhotla, H, Delaney, DD, Ward, M, Van, GF, Howard, JA, Hood, EE 2004Improved recovery of active recombinant laccase from maize seedAppl Microbiol Biotechnol63390397CrossRefPubMedGoogle Scholar
  3. Baker, JO, Adney, WS, Nieves, RA, Thomas, SR, Wilson, DB, Himel, ME 1994A new thermostable endoglucanase, Acidothermus cellulolyticus E1: synergism with Trichoderma reesei CBHI and comparison to thermomonospora fusca E15Appl Biochem Biotechnol45–46245256Google Scholar
  4. Barker, RF, Idler, KB, Thompson, DV, Kemp, JD 1983Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955Plant Mol Biol2335350CrossRefGoogle Scholar
  5. Bergquist, PL, Te’o, VS, Gibbs, MD, Curach, NC, Nevalainen, KM 2004Recombinant enzymes from thermophilic micro-organisms expressed in fungal hostsBiochem Soc Trans32293297CrossRefPubMedGoogle Scholar
  6. Biomass plan (2004) The US Department of Energy Biomass program-multi-year technical plan.Google Scholar
  7. Browning, KS, Lax, SR, Humphreys, J, Ravel, JM, Jobling, SA, Gehrke, L 1988Evidence that the 5’-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4GJ Biol Chem26396309634PubMedGoogle Scholar
  8. Chrispeels, MJ 1991Sorting of proteins in the secretory systemAnnu Rev Plant Physiol Plant Mol Biol422153CrossRefGoogle Scholar
  9. Comai, L, Moran, P, Maslyar, D 1990Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elementsPlant Mol Biol15373381CrossRefPubMedGoogle Scholar
  10. Conrad, U, Fiedler, U 1998Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activityPlant Mol Biol38101109CrossRefPubMedGoogle Scholar
  11. Cornelissen, BJ, Hooft Huijsduijnen, RA, Bol, JF 1986A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatinNature321531532CrossRefPubMedGoogle Scholar
  12. Dai, Z, Hooker, BS, Anderson, DB, Thomas, SR 2000aExpression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effectsTransgenic Res94354CrossRefGoogle Scholar
  13. Dai, Z, Hooker, BS, Anderson, DB, Thomas, SR 2000bImproved plant-based production of E1 endoglucanase using potato: expression optimization and tissue targetingMol Breed6277285CrossRefGoogle Scholar
  14. Dai, Z, Hooker, BS, Quesenberry, RD, Gao, J 1999Expression of Trichoderma reesei Exo-cellobiohydrolase I in transgenic tobacco leaves and calliAppl Biochem Biotechnol77–79689699CrossRefPubMedGoogle Scholar
  15. Dai, Z, An, G 1995Induction of nopaline synthase promoter activity by H2O2 has no direct correlation with salicylic acidPlant Physiol10911911197CrossRefPubMedGoogle Scholar
  16. Jaeger, G., Scheffer, S, Jacobs, A, Zambre, M, Zobell, O, Goossens, A, Depicker, A, Angenon, G 2002Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequencesNat Biotechnol2012651268CrossRefPubMedGoogle Scholar
  17. Denecke, J, Botterman, J, Deblaere, R 1990Protein secretion in plant cells can occur via a default pathwayPlant Cell25159CrossRefPubMedGoogle Scholar
  18. Divne, C, Stahlberg, J, Teeri, TT, Jones, TA 1998High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reeseiJ Mol Biol275309325CrossRefPubMedGoogle Scholar
  19. Fiedler, U, Phillips, J, Artsaenko, O, Conrad, U 1997Optimization of scFv antibody production in transgenic plantsImmunotechnol3205216CrossRefGoogle Scholar
  20. Gallie, DR, Sleat, DE, Watts, JW, Turner, PC, Wilson, TM 1987A comparison of eukaryotic viral 5’-leader sequences as enhancers of mRNA expression in vivoNucleic Acids Res1586938711PubMedGoogle Scholar
  21. Gallie, DR 1998Controlling gene expression in transgenicsCurr Opin Plant Biol1166172CrossRefPubMedGoogle Scholar
  22. Gallie, DR, Young, TE 1994The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expressionPlant Physiol106929939CrossRefPubMedGoogle Scholar
  23. Gittins, JR, Pellny, TK, Hiles, ER, Rosa, C, Biricolti, S, James, DJ 2000Transgene expression driven by heterologous ribulose-1, 5-bisphosphate carboxylase/oxygenase small-sub-unit gene promoters in the vegetative tissues of apple (Malus pumila mill.)Planta210232240PubMedGoogle Scholar
  24. Hann, LE, Gehrke, L 1995mRNAs containing the unstructured 5’ leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cellsJ Virol6949864993PubMedGoogle Scholar
  25. Herbers, K, Monke, G, Badur, R, Sonnewald, U 1995A thermostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily purifiedBio/technol136366CrossRefGoogle Scholar
  26. Hoekema, A, Hooykaas, PJ, Schilperoort, RA 1983A binary vector strategy based on seperation vir- and T-region of the Agrobacterium tumefaciens Ti-plasmidNature303179181CrossRefGoogle Scholar
  27. Holsters, M, de, WD, Depicker, A, Messens, E, Van, MM, Schell, J 1978ransfection and transformation of Agrobacterium tumefaciensMol Gen Genet163181187CrossRefPubMedGoogle Scholar
  28. Jensen, LG, Olsen, O, Kops, O, Wolf, N, Thomsen, KK, von Wettstein, D 1996Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germinationProc Natl Acad Sci U S A9334873491CrossRefPubMedGoogle Scholar
  29. Jobling, SA, Gehrke, L 1987Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequenceNature325622625CrossRefPubMedGoogle Scholar
  30. Kay, R, Chan, A, Daly, M, McPherson, J 1987Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genesScience23612991302Google Scholar
  31. Koziel, MG, Carozzi, NB, Desai, N 1996Optimizing expression of transgenes with an emphasis on post-transcriptional eventsPlant Mol Biol32393405CrossRefPubMedGoogle Scholar
  32. Laporte, MM, Galagan, JA, Prasch, AL, Vanderveer, PJ, Hanson, DT, Shewmaker, CK, Sharkey, TD 2001Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthasePlanta212817822CrossRefPubMedGoogle Scholar
  33. Liu, JH, Selinger, LB, Cheng, KJ, Beauchemin, KA, Moloney, MM 1997Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus Neocallimastix patriciarumMol Breed3463470CrossRefGoogle Scholar
  34. Manzara, T, Carrasco, P, Gruissem, W 1991Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene familyPlant Cell313051316CrossRefPubMedGoogle Scholar
  35. Matsuoka, K, Bassham, DC, Raikhel, NV, Nakamura, K 1995Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cellsJ Cell Biol13013071318CrossRefPubMedGoogle Scholar
  36. Matsuoka, K, Nakamura, K 1991Propeptide of a precursor to a plant vacuolar protein required for vacuolar targetingProc Natl Acad Sci U S A88834838PubMedGoogle Scholar
  37. Murashige, T, Skoog, F 1962A revised medium for rapid growth and bioassays with tobacco tissue culturesPhysiol Plant15473497Google Scholar
  38. Nagao, I, Obokata, J 2003A poly(U) motif in the 5’ untranslated region enhances the translational efficiency of β-glucuronidase mRNA in transgenic tobaccoPlant Sci165621626CrossRefGoogle Scholar
  39. Okamoto, T, Shimada, T, Hara-Nishimura, I, Nishimura, M, Minamikawa, T 2003C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidasePlant Physiol13218921900CrossRefPubMedGoogle Scholar
  40. Outchkourov, NS, Rogelj, B, Strukelj, B, Jongsma, MA 2003Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein productionPlant Physiol133379390CrossRefPubMedGoogle Scholar
  41. Outchkourov, NS, Peters, J, de, JJ, Rademakers, W, Jongsma, MA 2003The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plantsPlanta21610031012PubMedGoogle Scholar
  42. Park, JH, Oh, SA, Kim, YH, Woo, HR, Nam, HG 1998Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in ArabidopsisPlant Mol Biol37445454CrossRefPubMedGoogle Scholar
  43. Pauly, M, Andersen, LN, Kauppinen, S, Kofod, LV, York, WS, Albersheim, P, Darvill, A 1999A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzymeGlycobiology993100CrossRefPubMedGoogle Scholar
  44. Pichersky, E, Bernatzky, R, Tanksley, SD, Cashmore, AR 1986Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small sub-unit of ribulose-1,5-bisphosphate carboxylase/oxygenaseProc Natl Acad Sci U S A8338803884PubMedGoogle Scholar
  45. Sambrook, JF, Fritsch, EF, Maniatis, T 1989Molecular Cloning: A Laboratory ManualCold Spring Harbor Laboratory PressCold Spring Harbor, NewYorkGoogle Scholar
  46. Schouten, A, Roosien, J, Engelen, FA, Jong, GA, Borst-Vrenssen, AW, Zilverentant, JF, Bosch, D, Stiekema, WJ, Gommers, FJ, Schots, A, Bakker, J 1996The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobaccoPlant Mol Biol30781793CrossRefPubMedGoogle Scholar
  47. Sugita, M, Manzara, T, Pichersky, E, Cashmore, A, Gruissem, W 1987Genomic organization, sequence analysis and expression of all five genes encoding the small sub-unit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomatoMol Gen Genet209247256CrossRefPubMedGoogle Scholar
  48. Tucker, MP, Mohagheghi, A, JGrohman, K, Himmel, ME 1989Ultra-thermostable cellulases from Acidothermus cellulolyticus: comparison of temerature optima with previously reported cellulasesBiotechnology7817820CrossRefGoogle Scholar
  49. Verwoerd, TC, Paridon, PA, Ooyen, AJ, Lent, JW, Hoekema, A, Pen, J 1995Stable accumulation of Aspergillus niger phytase in transgenic tobacco leavesPlant Physiol10911991205CrossRefPubMedGoogle Scholar
  50. Vitale, A, Denecke, J 1999The endoplasmic reticulum-gateway of the secretory pathwayPlant Cell11615628CrossRefPubMedGoogle Scholar
  51. Ossowski, I, Terri, T, Kalkkinen, N, Oker-Blom, C 1997Expression of a fungal cellobiohydrolase in insect cellsBiochem Biophys Res Com2332529CrossRefPubMedGoogle Scholar
  52. Ziegelhoffer, T, Raasch, JA, Austin-Phillips, S 2001Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobaccoMol Breed8147158CrossRefGoogle Scholar
  53. Ziegler, MT, Thomas, SR, Danna, KJ 2001Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leavesMol Breed63746CrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Ziyu Dai
    • 1
    Email author
  • Brian S. Hooker
    • 1
  • Ryan D. Quesenberry
    • 1
  • Steven R. Thomas
    • 2
  1. 1.Chemical and Biological Processing Development Group, Process Science and Engineering DivisionPacific Northwest National LaboratoryRichlandUSA
  2. 2.National Renewable Energy LaboratoryGoldenUSA

Personalised recommendations