Advertisement

Biotechnology Letters

, 33:1797 | Cite as

Expressions of thermostable bacterial cellulases in tobacco plant

  • Xi-ran Jiang
  • Xiao-ya Zhou
  • Wen-yan Jiang
  • Xiao-rong Gao
  • Wen-li LiEmail author
Original Research Paper

Abstract

An economical method for the conversion of lignocellulosic biomass is to use plants as bioreactors for cellulases production. Two bacterial thermostable cellulases (E2 and E3) and a E3-E2 fusion form were expressed in tobacco, driven by a double 35S promoter and 5′ TEV-UTL. The enzymes were targeted to the apoplast and cytosol via 5′ signal peptides and 3′ retention signal peptides, respectively, and all showed functional activities. All transgenic plants exhibited normal growth compared to wild type. Transgenic plants that expressed apoplast-localized E2 had the highest average activity, about 1.5 and 3 times higher than those expressed ER-localized and cytosolic E2, respectively. Effect of subcellular compartment localization was due primarily to post-transcriptional modification, since mRNA abundances were similar despite the range of cellulase activities obtained. The recombinant cellulases exhibited good thermostability below 65°C. After storing for 3 days at −20 and 28°C, the enzymes lost nearly 20 and 80% of activity, respectively. The results suggested a potential application for heterologous expression of cellulases in plant for biomass conversion.

Keywords

Biomass Cellulase Sub-cellular targeting Transgenic tobacco 

Abbreviations

35S

Cauliflower Mosaic Virus 35s

TEV

Tobacco etch virus

UTL

Untranslated leader

CALSP

Tobacco calreticulin signal peptide

MUC

4-Methylumbelliferyl-β-d-cellobioside

MU

4-Methylumbelliferone

Notes

Acknowledgments

The work was supported by “the Fundamental Research Funds for the Central Universities” (DUT10JN01) and China Environmental Protection Foundation Liaoning Representative Office. Liaoning environmental scientific research “123” fund project (CEPF2008-123-2-11). We thank Alan K Chang (Dalian University of Technology) for helpful discussion and for revising the manuscript.

Supplementary material

10529_2011_642_MOESM1_ESM.doc (27 kb)
Supplementary material 1 (DOC 27 kb)
10529_2011_642_MOESM2_ESM.doc (33 kb)
Supplementary material 2 (DOC 33 kb)

References

  1. Biswas G, Ransom C, Sticklen M (2006) Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize plants. Plant Sci 171:617–623CrossRefGoogle Scholar
  2. Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38:101–109PubMedCrossRefGoogle Scholar
  3. Dai Z, Hooker BS, Anderson DB, Thomas SR (2000) Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects. Transgenic Res 9:43–54PubMedCrossRefGoogle Scholar
  4. Dai Z, Hooker BS, Quesenberry RD, Thomas SR (2005) Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification. Transgenic Res 14:627–643PubMedCrossRefGoogle Scholar
  5. Jiang XR, Li WL (2009) Research progress in genetic engineering for cellulosic ethanol. China Biotechnol 29:127–133Google Scholar
  6. Jiang XR, Liu JW, Li WL (2010) The progress in thermophile research and its application prospect in cellulosic ethanol. Life Sci Res 5:449–455Google Scholar
  7. Jung S, Kim S, Bae H, Lim HS, Bae HJ (2010) Expression of thermostable bacterial β-glucosidase (BglB) in transgenic tobacco plants. Bioresour Technol 101:7144–7150CrossRefGoogle Scholar
  8. Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292PubMedCrossRefGoogle Scholar
  9. Pang Z, Dong Z, Liang J, Chen S, Wei P, Huang R (2005) Study on cellulase properties of Thermophilic actinomycete GPL1. Mod Food Sci Technol 22:20–23Google Scholar
  10. Wilson DB (2004) Studies of Thermobifida fusca plant cell wall degrading enzymes. Chem Rec 4:72–82PubMedCrossRefGoogle Scholar
  11. Youm JW, Kim H, Han JH, Jang CH, Ha HJ, Mook-Jung I, Jeon JH, Choi CY, Kim YH, Kim HS, Joung H (2005) Transgenic potato expressing Aβ reduce Aβ burden in Alzheimer’s disease mouse model. FEBS Lett 579:6737–6744PubMedCrossRefGoogle Scholar
  12. 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 5:309–318CrossRefGoogle Scholar
  13. Ziegelhoffer T, Raasch JA, Austin-Phillips S (2001) Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobacco. Mol Breed 8:147–158CrossRefGoogle Scholar
  14. Ziegler MT, Thomas SR, Danna KJ (2000) Accumulation of a thermostable endo-1, 4-β-d-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6:37–46CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Xi-ran Jiang
    • 1
  • Xiao-ya Zhou
    • 1
  • Wen-yan Jiang
    • 2
  • Xiao-rong Gao
    • 1
  • Wen-li Li
    • 1
    Email author
  1. 1.School of Life Science and BiotechnologyDalian University of TechnologyDalianPeople’s Republic of China
  2. 2.Department of PsychiatryThe First Affiliated Hospital, China Medical UniversityShenyangPeople’s Republic of China

Personalised recommendations