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Plant-Based Cellulase Assay Systems as Alternatives for Synthetic Substrates

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Abstract

Dissociative enzymes such as cellulases are greatly desired for a variety of applications in the food, fuel, and fiber industries. Cellulases and other cell wall–degrading enzymes are currently being engineered with improved traits for application in the breakdown of lignocellulosic biomass. Biochemical assays using these “designer” enzymes have traditionally been carried out using synthetic substrates such as crystalline bacterial microcellulose (BMCC). However, the use of synthetic substrates may not reflect the actual action of these cellulases on real plant biomass. We examined the potential of suspension cell walls from several plant species as possible alternatives for synthetic cellulose substrates. Suspension cells grow synchronously; hence, their cell walls are more uniform than those derived from mature plants. This work will help to establish a new assay system that is more genuine than using synthetic substrates. In addition to this, we have demonstrated that it is feasible to produce cellulases inexpensively and at high concentrations and activities in plants using a recombinant plant virus expression system. Our long-term goals are to use this system to develop tailored cocktails of cellulases that have been engineered to function optimally for specific tasks (i.e., the conversion of biomass into biofuel or the enhancement of nutrients available in livestock feed). The broad impact would be to provide a facile and economic system for generating industrial enzymes that offer green solutions to valorize biomass in industrialized communities and specifically in developing countries.

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References

  1. Bhat, M. (2000). Cellulases and related enzymes in biotechnology. Biotechnology Advances, 18(5), 355–383.

    Article  CAS  Google Scholar 

  2. Park, S. H., Ong, R. G., & Sticklen, M. (2016 Jun). Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production. Plant Biotechnology Journal, 14(6), 1329–1344.

    Article  CAS  Google Scholar 

  3. Ruth, M.; Mai, T.; Newes, E.; Aden, A.; Warner, E.; Uriarte, C; Inman, D.; Simpkins, T.; Argo, A. (0DUFK 2013). Projected biomass utilization for fuels and power in a mature market. Transportation Energy Futures Series. Prepared for the U.S. Department of Energy by National Renewable Energy Laboratory, Golden, CO. DOE/GO-102013-3707. 153 pp.

  4. Johnson, E. (2016). Integrated enzyme production lowers the cost of cellulosic ethanol. Biofuels Bioproducts and Biorefining, 10(2), 164–174.

    Article  CAS  Google Scholar 

  5. Wilson, D. B. (2004). Studies of Thermobifida fusca plant cell wall degrading enzymes. Chemical Record, 4(2), 72–82.

    Article  CAS  Google Scholar 

  6. Sticklen, M. B. (2008). Plant genetic engineering for biofuel production: Towards affordable cellulosic ethanol. Nature Reviews. Genetics, 9(6), 433–443.

    Article  CAS  Google Scholar 

  7. Kostylev, M., & Wilson, D. (2012). Synergistic interactions in cellulose hydrolysis. Biofuels, 3(1), 61–70.

    Article  CAS  Google Scholar 

  8. Jalak, J., Kurašin, M., Teugjas, H., & Väljamäe, P. (2012). Endo-exo synergism in cellulose hydrolysis revisited. The Journal of Biological Chemistry, 287(34), 28802–28815.

    Article  CAS  Google Scholar 

  9. Santos, R. B., Abranches, R., Fischer, R., Sack, M., & Holland, T. (2016). Putting the spotlight back on plant suspension cultures. Frontiers in Plant Science, 7, 297. Published online 2016 Mar 11. https://doi.org/10.3389/fpls.2016.00297.

    Article  Google Scholar 

  10. Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., & Fischer, R. (2003). Molecular farming in plants: Host ¨ systems and expression technology. Trends in Biotechnology, 1, 570–578.

    Article  Google Scholar 

  11. Wong-Arce, A., González-Ortega, O., & Rosales-Mendoza, S. (2017). Plant-made vaccines in the fight against cancer. Trends in Biotechnology, 35(3), 241–256.

    Article  CAS  Google Scholar 

  12. Holtz, B. R., Berquist, B. R., Bennett, L. D., Kommineni, V. J. M., Munigunti, R. K., White, E. L., Wilkerson, D. C., Wong, K. Y. I., Ly, L. H., & Marcel, S. (2015). Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals. Plant Biotechnology Journal, 13(8), 1180–1190.

    Article  CAS  Google Scholar 

  13. Hefferon, K. (2017). Plant virus expression vectors: A powerhouse for global health. Biomedicines, 5(3), 44. Published online 2017 Jul 30. https://doi.org/10.3390/biomedicines5030044.

    Article  CAS  Google Scholar 

  14. Tschofen, M., Knopp, D., Hood, E., & Stoger, E. (2016). Plant molecular farming: Much more than medicines annual rev. Analytical Chemistry, 9, 271–294.

    Google Scholar 

  15. Wu, J., Wang, M., Zhang, H., & Liu, R. (2015). Extreme thermophilic enzyme CelB-m efficiently degrades the cellulose in transgenic Arabidopsis thaliana. Applied Biochemistry and Biotechnology, 177(2), 362–372.

    Article  CAS  Google Scholar 

  16. Tomassetti, S., Pontiggia, D., Verrascina, I., Reca, I. B., Francocci, F., Salvi, G., Cervone, F., & Ferrari, S. (2015). Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth. Phytochemistry, 112, 221–230.

    Article  CAS  Google Scholar 

  17. Ransom, C., Balan, V., Biswas, G., Dale, B., Crockett, E., & Sticklen, M. (2007). Heterologous Acidothermus cellulolyticus 1,4-beta-endoglucanase E1 produced within the corn biomass converts corn stover into glucose. Applied Biochemistry and Biotechnology, 137–140(1–12), 207–219.

    Google Scholar 

  18. Min, D., Li, Q., Jameel, H., Chiang, V., & Chang, H. M. (2012). The cellulase-mediated saccharification on wood derived from transgenic low-lignin lines of black cottonwood (Populus trichocarpa). Applied Biochemistry and Biotechnology, 168(4), 947–955.

    Article  CAS  Google Scholar 

  19. Jung, S.-K., Parisutham, V., Jeong, S. H., & Lee, S. K. (2012). Heterologous expression of plant cell wall degrading enzymes for effective production of cellulosic biofuels. Journal of Biomedicine & Biotechnology, 405842.

  20. Jiang, X. R., Zhou, X. Y., Jiang, W. Y., Gao, X. R., & Li, W. L. (2011). Expressions of thermostable bacterial cellulases in tobacco plant. Biotechnology Letters, 33(9), 1797–1803.

    Article  CAS  Google Scholar 

  21. Klinger, J., Fischer, R., & Commandeur, U. (2015). Comparison of Thermobifida fusca cellulases expressed in Escherichia coli and indicates advantages of the plant system for the expression of bacterial cellulases. Frontiers in Plant Science, 6, 1047.

    Article  Google Scholar 

  22. Hood EE. Plant-based biofuels F1000Research 2016, 5(F1000 Faculty Rev):185.

  23. Kostylev, M., Wilson, D. A distinct model of synergism between a processive endocellulase (TfCel9A) and an exocellulase (TfCel48A) from Thermobifida fusca. January 2014 Volume 80 Number 1 Applied and Environmental Microbiology p. 339–344.

  24. Kruer-Zerhusen N, Cantero-Tubilla B, Wilson D. Characterization of cellulose crystallinity after enzymatic treatment using Fourier transform infrared spectroscopy (FTIR). Cellulose. 2017.

  25. King, B. C., Donnelly, M. K., Bergstrom, G. C., Walker, L. P., & Gibson, D. M. (2009). An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts. Biotechnology and Bioengineering, 102(4), 1033–1044. https://doi.org/10.1002/bit.22151.

    Article  CAS  Google Scholar 

  26. Cruys-Bagger, N., Elmerdahl, J., Praestgaard, E., Tatsumi, H., Spodsberg, N., Borch, K., & Westh, P. (2012). Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A. The Journal of Biological Chemistry, 287(22), 18451–18458. https://doi.org/10.1074/jbc.M111.334946 Epub 2012 Apr 9.

    Article  CAS  Google Scholar 

  27. Hefferon, K. L., & Dugdale, B. G. (2003). Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication. Journal of General Virology, 84(12), 3465–3472.

    Article  CAS  Google Scholar 

  28. Hefferon, K. L., & Fan, Y. (2004). Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system. Vaccine., 23(3), 404–410.

    Article  CAS  Google Scholar 

  29. Schmidt, J. A., McGrath, J. M., Hanson, M. R., Long, S. P., & Ahner, B. A. (2019). Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts. Nature Plants, 5(7), 715–721. https://doi.org/10.1038/s41477-019-0467-z Epub 2019 Jul 8.

    Article  CAS  Google Scholar 

  30. Nakahira, Y., Ishikawa, K., Tanaka, K., Tozawa, Y., & Shiina, T. (2013). Overproduction of hyperthermostable β-1,4-endoglucanase from the archaeon Pyrococcus horikoshii by tobacco chloroplast engineering. Bioscience, Biotechnology, and Biochemistry, 77(10), 2140–2143.

    Article  CAS  Google Scholar 

  31. Longoni, P., Leelavathi, S., Doria, E., Reddy, V. S., & Cella, R. (2015). Production by tobacco transplastomic plants of recombinant fungal and bacterial cell-wall degrading enzymes to be used for cellulosic biomass saccharification. BioMed Research International, 289759.

  32. Giritch, A., Klimyuk, V., & Gleba, Y. (2017). 125 years of virology and ascent of biotechnologies based on viral expression. Tsitologiia i Genetika, 51(2), 19–39.

    CAS  Google Scholar 

  33. Song, E. G., & Ryu, K. H. (2017). A pepper mottle virus-based vector enables systemic expression of endoglucanase D in non-transgenic plants. Archives of Virology, 162(12), 3717–3726.

    Article  CAS  Google Scholar 

  34. Hood E, Requesens D. Commercial plant-produced recombinant cellulases for biomass conversion. Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 68) 2014. , pp. 231–46.

  35. Zhang, S., Barr, B. K., & Wilson, D. B. (2000). Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A. European Journal of Biochemistry, 267(1), 244–252.

    Article  CAS  Google Scholar 

  36. Li, Q., Song, J., Peng, S., Wang, J. P., & Qu, G. Z. (2014). Plant biotechnology for lignocellulosic biofuel production. Plant Biotechnology Journal, 12(9), 1174–1192.

    Article  CAS  Google Scholar 

  37. Kumari, U., Singh, R., Ray, T., Rana, S., Saha, P., Malhotra, K., & Daniell, H. (2019). Validation of leaf enzymes in the detergent and textile industries: Launching of a new platform technology. Plant Biotechnology Journal, 17(6), 1167–1182. https://doi.org/10.1111/pbi.13122 Epub 2019 Apr 23.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14850, USA

    Kathleen Hefferon & David W. Wilson

  2. Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14850, USA

    Borja Cantero-Tubilla

  3. Cell and Systems Biology, University of Toronto, Toronto, Canada

    Uzma Badar

Authors
  1. Kathleen Hefferon
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  2. Borja Cantero-Tubilla
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  3. Uzma Badar
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  4. David W. Wilson
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Correspondence to Kathleen Hefferon.

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Hefferon, K., Cantero-Tubilla, B., Badar , U. et al. Plant-Based Cellulase Assay Systems as Alternatives for Synthetic Substrates. Appl Biochem Biotechnol 192, 1318–1330 (2020). https://doi.org/10.1007/s12010-020-03395-7

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