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Recombinant Cellulase Accumulation in the Leaves of Mature, Vegetatively Propagated Transgenic Sugarcane

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Abstract

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

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Acknowledgments

The authors are grateful to Paul Oeller, Jason Nichols, Kerry Kaffall, and Sergio Arellano at Syngenta Biotechnology Incorporated for providing anti-cellulase antibodies, denatured positive control proteins for western blotting, and for helpful discussions. The authors thank William Doherty for providing valuable comments on the manuscript. The authors also thank the staff at the Queensland Crop Development Facility (Department of Agriculture, Fisheries and Forestry, Queensland State Government) for their assistance with the growth of transgenic sugarcane. Mark Harrison was the recipient of a Smart State Fellowship from the Queensland State Government. The Queensland Crop Development Facility was funded by a grant from the Smart State Research Facilities Fund Scheme of the Queensland State Government. The Syngenta Centre for Sugarcane Biofuels Development is supported by Syngenta Biotechnology Incorporated, Queensland University of Technology, Leaf Energy Ltd., and by a grant from the National and International Research Alliances Program of the Queensland State Government.

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Author notes

  1. R. Jason Geijskes

    Present address: Syngenta Proteçâo de Cultivos Ltda, Rod. BR 452 Km 142, Uberlândia, MG, CEP 38400-974, Brazil

  2. Anthony Palupe

    Present address: Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD, 4001, Australia

  3. Manuel B. Sainz

    Present address: Syngenta Proteção de Cultivos Ltda, Av Maurílio Biagi 800, 9º andar, Ribeirão Prêto, São Paulo, CEP 14020-750, Brazil

Authors and Affiliations

  1. Syngenta Centre for Sugarcane Biofuels Development, Centre for Tropical Crops and Biocommodities, Queensland University of Technology, 2 George Street, Brisbane, QLD, 4001, Australia

    Mark D. Harrison, R. Jason Geijskes, Robyn Lloyd, Anthony Palupe, Manuel B. Sainz & James L. Dale

  2. Syngenta Biotechnology Inc., 3054 East Cornwallis Road, Research Triangle Park, NC, 27709-2257, USA

    Stacy Miles

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  1. Mark D. Harrison
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Correspondence to Mark D. Harrison.

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Harrison, M.D., Geijskes, R.J., Lloyd, R. et al. Recombinant Cellulase Accumulation in the Leaves of Mature, Vegetatively Propagated Transgenic Sugarcane. Mol Biotechnol 56, 795–802 (2014). https://doi.org/10.1007/s12033-014-9758-9

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  • DOI: https://doi.org/10.1007/s12033-014-9758-9

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