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

Plant Molecular Biology volume 83, pages 317–328 (2013)Cite this article

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.

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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.

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Authors and Affiliations

  1. Department of Forest Products and Technology, Chonnam National University, Kwangju, 500-757, Republic of Korea

    Sera Jung

  2. Bio-energy Research Institute, Chonnam National University, Kwangju, 500-757, Republic of Korea

    Dae-Seok Lee & Yeon-Ok Kim

  3. Biotechnology Research Center, School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI, 49931, USA

    Chandrashekhar P. Joshi

  4. Department of Bioenergy Science and Technology, Chonnam National University, Kwangju, 500-757, Republic of Korea

    Hyeun-Jong Bae

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  1. Sera Jung
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  2. Dae-Seok Lee
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  4. Chandrashekhar P. Joshi
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  5. Hyeun-Jong Bae
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Correspondence to Hyeun-Jong Bae.

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Jung, S., Lee, DS., Kim, YO. et al. Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5′ amplification promoting sequence. Plant Mol Biol 83, 317–328 (2013). https://doi.org/10.1007/s11103-013-0088-2

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  • DOI: https://doi.org/10.1007/s11103-013-0088-2

Keywords

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

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