Plant Molecular Biology

, Volume 83, Issue 4–5, pp 317–328 | Cite as

Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5′ amplification promoting sequence

  • Sera Jung
  • Dae-Seok Lee
  • Yeon-Ok Kim
  • Chandrashekhar P. Joshi
  • Hyeun-Jong Bae
Article
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Accepted:

Abstract

Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.

Keywords

Molecular farming Transgenic plant Promoter engineering Bacterial cellulase Thermotoga maritima Beta-glucosidase 
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Notes

Acknowledgments

This work was supported by Priority Research Centers Program (2010-0020141), and WCU (World Class University) project (R31-2009-000-20025-0) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology to H.-J. Bae.

Supplementary material

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Supplementary material 1 (TIFF 2770 kb)
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Supplementary material 2 (TIFF 814 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Sera Jung
    • 1
  • Dae-Seok Lee
    • 2
  • Yeon-Ok Kim
    • 2
  • Chandrashekhar P. Joshi
    • 3
  • Hyeun-Jong Bae
    • 4
  1. 1.Department of Forest Products and TechnologyChonnam National UniversityKwangjuRepublic of Korea
  2. 2.Bio-energy Research InstituteChonnam National UniversityKwangjuRepublic of Korea
  3. 3.Biotechnology Research Center, School of Forest Resources and Environmental ScienceMichigan Technological UniversityHoughtonUSA
  4. 4.Department of Bioenergy Science and TechnologyChonnam National UniversityKwangjuRepublic of Korea

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