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

Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Springer Netherlands
Copyright
Copyright © 2013 by Springer Science+Business Media Dordrecht
Subject
Life Sciences; Plant Sciences; Biochemistry, general; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-013-0088-2
Publisher site
See Article on Publisher Site

References

  • Second-generation biofuels and local bioenergy systems
    Antizar-Ladislao, B; Torrion-Gomez, JL
  • Production of a recombinant xylanase in plants and its potential for pulp biobleaching applications
    Bae, H-J; Kim, HJ; Kim, YS
  • Commercializing lignocellulosic bioethanol: technology bottlenecks and possible remedies
    Banerjee, S; Mudliar, S; Sen, R; Giri, B; Satpute, D; Chakrabarti, T; Pandey, RA
  • Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize plants
    Biswas, GCG; Ransom, C; Sticklen, M
  • Constitutive, light-responsive and circadian clock-responsive factors compete for the different I box elements in plant light-regulated promoters
    Borello, U; Ceccarelli, E; Giovanni, G
  • Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
    Clough, SJ; Bent, AF
  • Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli
    Dai, Z; Hooker, BS; Quesenberry, RD; Gao, J
  • High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions
    Gray, BN; Ahner, BA; Hanson, MR

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