Plant Cell Wall Modificationfor the Production of Biofuel

By Ben Merritt

Why Modify the Cell Wall?

• We are running out of unsustainable fuel sources

• Plant cell wall contains “locked up” source of carbon

Composition of Plant Cell Wall

• 3 layers • Middle Lamella (outermost from cytoplasm;

connects cells); made of pectin• Primary; made of pectin, cellulose,

hemicellulose, proteins• Secondary; made of cellulose, hemicellulose,

lignin (only in mature cells)

Cellulose (Cellubiose)

Cellulose (Crystalline)

Cellulose Arrangement

Hemicellulose vs. Cellulose

Hemicellulose (Xylan), continued

Lignin

Biomass Production

Yeast Fermentation

Perfect Biomass Plant?

• High Cellulose content

• Low lignin content

• Low ash content (unusable inorganics)

First/Second Generation Biofuel

• 1st = using edible sugars (sucrose) from food crops for fuel production

• 2nd = using non-edible, structural polysaccharides for fuel production

2nd Generation Biofuels

• 1.5 billion tons of unused plant material each year

• Is estimated to be equivalent to total oil imports (442 billion liters of EtOH)

Acid Growth – Natural Cell Wall Modification

• Mediated by auxin (cell elongation)

• Physical teasing apart of cell wall

• Expansins, endoglucanases spread apart and degrade parts

Enhance Efficiency of Reclaim for Ethanol

• Increase cellulose/cell wall density

• Remove unwanteds (hemicellulose, lignin)

• Incorporate soluble monomers (makes processing easier)

• Utilize glucanases (increases growth)

• Destroy lignin-cellulose bonds (decreases lignin)

Chloroplast-Derived Enzyme Cocktail

• One team inserted:• Endo/exoglucanase or lipase from bacteria• Pectate Lyases• Cutinase• Endoglucanases• Swollenin• Xylanase• Acetyl Xylan Esterase• Beta glucosidase

“Recalcitrance of Lignocellulosic Material”

• Cellulose evolved to be tough

• Microbes are the main cell wall metabolizers (bacteria, fungi)

• Endoglucanases also found in guts of termites, cockraoches, grass carps, blue mussel

Acetobacter xylinum

References• Abramson, M., O. Shoseyov, S. Hirsch, and Z. Shani. “Genetic Modifications of Plant Cell Walls to Increase Biomass and Bioethanol Production.” Published in Advanced Biofuels and

Bioproducts. New York 2013.• • Buanafina, M.M., T. Langdon, B. Hauck, S. Dalton, E. Timms-Taravella, and P. Morris. “Targeting Expression of a Fungal Ferulic Acid Esterase to the Apoplast, Endoplasmic Reticulum or

Golgi can Disrupt Feruloylation of the Growing Cell Wall and Increase the Biodegradability of Tall Fescue (Festuca arundinacea). Plant Biotecnol. (8):316-331. 2010.• • Delmer, D.P. and Y. Amor. “Cellulose Biosynthesis.” The Plant Cell. (7):987-1000. July 1995.• • Fu, C., R. Sunkar, C. Zhou, H. Shen, J.Y. Zhang, J. Matts, J. Wolf, D.G. Mann, C.N. Stewart, Y. Tang et al. “Overexpression of miR156 in Switchgrass (Panicum virgatum L. Results in Various

Morphological Alterations and Leads to Improved Biomass Production. Plant Biotechnology. (10):443-452. 2012.• • FuturaGene. “FuturaGene Submits Genetically Modified Eucalyptus for Commercial Approval.” < http://www.futuragene.com/H421-Dossier-submission-website-May-2014-English-FINAL-

VERSION.pdf > May 2014.• • Furtado, A., J.S. Lupoi, N.V. Hoang, A. Healey, S. Singh, B.A. Simmons, and R.J. Henry. “Modifying Plants for Biofuel and Biomaterial Production.” Plant Biotechnology. (12):1246-1258.

October 2014.• • Hu, W.J., S.A. Harding, J. Lung, J.L. Popko, J. Ralph, D.D. Stokke, C.J. Tsai, and V.L. Chiang. “Repression of Lignin Biosynthesis Promotes Cellulose Accumulation and Growth in Transgenic

Trees.” Nat. Biotechnol. (17):808-812. 1999.• • Joshi, C.P., S. Thammannagowda, T. Fujino, J.Q. Gou, U. Avci, C.H. Haigler, L.M. McDonnell, S.D. Mansfield, B. Mengesha, N.C. Carpita, et al. “Perturbation of Wood Cellulose Synthesis

Causes Pleiotropic Effects in Transgenic Aspen. Mol. Plant. (4):331-345. 2011.• • Jung, J.H., W.M. Fouad, W. Vermerris, M. Gallo, and F. Altpeter. “RNAi Suppression of Lignin Biosynthesis is Sugarcane Reduces Recalcitrance for Biofuel Production from Lignocellulosic

Biomass.” Plant Biotechnol. (10):1067-1076. 2012.• • Lee, C., Q. Teng, W. Huang, R. Zhong, and Z.H. Ye. “Down-Regulation of PoGT47c Expression in Poplar Results in a Reduced Glucuronoxylan Content and an Increased Wood Digestibility by

Cellulase.” Plant Cell Physiol. (50):1075-1089. 2009.• • Levy, I., Z. Shani, and O. Shoseyov. “Modification of Polysaccharides and Plant Cell Wall by Endo-1,4-ß-glucanase and Cellulose-Binding Domains.” Biomolecular Engineering. (19):17-30.

March 2002.• • Park, Y.W., K. Baba, Y. Furuta, I. Iida, and K. Sameshima. “Enhancement of Growth and Cellulose Accumulation by Overexpression of Xyloglucanase in Poplar.” FEBS Lett. (564):183-187.

(2004). • • Rayle, D.L. and R.E. Cleland. “The Acid Growth Theory of Auxin-Induced Cell Elongation is Alive and Well.” Plant Physiol. (99):1271-1274. April 1992.• • Sahoo, D.K., J. Stork, S. Debolt, and I.B. Maiti. “Manipulating Cellulose Biosynthesis by Expression of Mutant Arabidopsis proM24::CESA(ixr1-2) Gene in Transgenic Tobacco.” Plant

Biotechnology. (11):362-372. 2013.• • Somerville, C. and J Milne. “Genetic Modification of Plant Cell Walls for Enhanced Biomass Production and Utilization.” GCEP Technical Report. 2005.• • Verma, D., A. Kanagaraj, S. Jin, N.D. Singh, P.E. Kolattukudy, and H. Daniell. “Chloroplast-Derived Enzyme Cocktails Hydrolyse Lignocellulosic Biomass and Release Fermentable Sugars.”

Plant Biotechnol. 8(3):332-350. April 2010.