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In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane

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Abstract

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.

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Acknowledgements

We would like to thank USDA-NIFA (Grant #2009-10001-05117) and Syngenta Biotechnology Inc. for their financial support of this research, Dr. Rob Gilbert University of Florida-IFAS, EREC, Belle Glade FL for providing donor plants of sugarcane cultivar CP88-1762 and Sun Gro Horticulture, Apopka, FL for donation of the Fafard #2 potting mix.

Author contributions

F.A., M.G. and J.F.P. conceived the experiments. F.A., J.Y.K. and J.F.P. designed the experiments. J.Y.K. constructed the plasmids, generated transgenic plants, completed molecular analysis of transgenic plants, analysis of Xyl10B activity, specific activity and total yield from different tissues and greenhouse propagation of transgenic plants (data shown in Figs. 1, 2 and Supplementary Fig. 1, Tables 1, 2, Supplementary Table1), G.N. and J.Y.K. analyzed the depolymerization of sugarcane xylan by Xyl10B, α-glucuronidase (AguA) and α-arabinofuranosidase (AbfA) (data shown in Fig. 3), J.D.R. generated the temperature response data of in planta produced Xyl10B shown in Fig. 4. J.Y.K., J.F.P. and F.A. wrote the manuscript. All authors read and approved the final manuscript.

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  1. Jae Yoon Kim

    Present address: Division of Biotechnology, Korea University, Seongbuk-Gu, Seoul, 02841, Republic of Korea

  2. Maria Gallo

    Present address: Delaware Valley University, Doylestown, PA, USA

Authors and Affiliations

  1. Plant Molecular and Cellular Biology Program, Agronomy Department, Genetics Institute, University of Florida - IFAS, Gainesville, FL, USA

    Jae Yoon Kim, Maria Gallo & Fredy Altpeter

  2. Department of Microbiology and Cell Science, University of Florida - IFAS, Gainesville, FL, USA

    Guang Nong, John D. Rice & James F. Preston

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Kim, J.Y., Nong, G., Rice, J.D. et al. In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane. Plant Mol Biol 93, 465–478 (2017). https://doi.org/10.1007/s11103-016-0573-5

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