1.
Applications of Biotechnology to Improve Energy Crops for Biofuel Production<br />David J. Glass, Ph.D.<br />D. Glass Associates, Inc.<br />World Biofuels Markets, Rotterdam<br />22 March 2011<br />

2.
Overview of Technology Strategies<br />

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Biotechnologies Applicable to Energy Crops<br />Classical plant breeding<br />Recombinant DNA<br />Agrobacterium-mediated transformation<br />Electroporation, “Gene Gun”<br />Synthetic biology<br />Genome sequencing<br />Plant breeding aided by genomics<br />Most commercially important plants can be genetically engineered, and genome sequences of many species are becoming available.<br />

4.
Energy Crop Genetic Modification Strategies<br />Enhancing substrate concentration <br />Engineer oilseed crops to have altered or enhanced lipid content<br />Engineer cellulosic crops to increase polysaccharide levels<br />Enhancing feedstock digestibility <br />Improve or optimize enzymatic degradation of cellulosic feedstock<br />Decrease or modify concentrations of lignins, other recalcitrant compounds<br />

5.
Energy Crop Genetic Modification Strategies<br />Increase plant biomass or crop yield<br />increasing plant growth rates<br />insect or herbicide resistance<br />drought tolerance<br />Plant genomics to aid breeding,genetic engineering<br />Use genetically modified plants to manufacture industrial enzymes<br />

6.
Companies Developing Modified Plants for Biofuels<br />Abba Gaia<br />Agragen<br />Agrisoma<br />Agrivida <br />ArborGen <br />CanaVialis S.A<br />Catchlight Energy<br />Ceres <br />Chromatin<br />Edenspace Systems<br />Evogene<br />Farmacule BioIndustries <br />FuturaGene<br />Iden Biotechnology<br />Kaiima<br />Life Technologies<br />Mendel Biotechnology<br />Metabolix <br />Naturally Scientific<br />RahanMeristem<br />SG Biofuels <br />Syngenta<br />Targeted Growth <br />

7.
Strategies to Enhance Lipid Content in Energy Crops<br />

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Enhancing Lipid Content<br />Enhance fatty acid biosynthesis<br />Overexpress ACCase <br />Enhance triacylglycerol synthesis<br />Overexpress DGAT, other pathway enzymes<br />

9.
Enhancing Lipid Content<br />Plant Biotechnol J. 2009 Sep;7(7):694-703.<br />Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways.<br />Slocombe SP, Cornah J, Pinfield-Wells H, Soady K, Zhang Q, Gilday A, Dyer JM, Graham IA<br />Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York, UK.<br />Plant Biotechnol J. 2010 Apr;8(3):277-87. Epub 2009 Dec 23.<br />Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. <br />Andrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M, Golovkin M, Koprowski H<br />Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, PA, USA.<br />

10.
Companies Enhancing Lipid Concentrations<br />Agragen: Camelina<br />Agrisoma: BrassicaandJatropha<br />Evogene: canola, soybean, others<br />Kaiima: castor beans (non-GMO)<br />Metabolix: oil crops<br />RahanMeristem: Jatropha, castor beans<br />SG Biofuels: Jatropha<br />Targeted Growth: Camelina, canola, others<br />

11.
Strategies to Enhance Polysaccharide Content in Energy Crops<br />

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Enhancing Carbohydrate, Polysaccharide Content<br /><ul><li>Increase concentration of glucose, other sugars

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Hexose, sucrose transporters

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Glucose signaling

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Increase cellulose, hemicellulose content in cell walls

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Cellulose synthase

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Glucosyltransferase

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Enhance starch biosynthesis

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Starch synthases

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Branching, debranching enzymes</li></ul>Source: Sticklen (2008) Nature Reviews Genetics 9:433-443<br />

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Companies Pursuing Enhanced Polysaccharide Content<br />Chromatin: switchgrass, Miscanthus, sorghum<br />FuturaGene: switchgrass, Miscanthus, hybrid poplar and willow <br />Mendel Biotechnology:grasses, others<br />Targeted Growth: corn<br />

22.
Strategies to Express Degradative Enzymes in Energy Crops<br />

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Expression of Degradative Enzymes<br />Express thermostable amylase in plant tissue, to enhance starch breakdown.<br />

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Expression of Degradative Enzymes<br />Express enzymes to enhance breakdown of hemicellulose.<br />

25.
Expression of Degradative Enzymes<br />Edenspace Systems: Express cellulases to enhance cellulose breakdown <br /><ul><li>Agrivida: Timed, inducible expression of cell-wall degrading enzymes</li></ul>Agrivida, Inc. © 2011<br />dormant enzyme<br />crop harvest and<br />enzyme activation<br />activated enzyme<br />

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Companies Expressing Degradative Enzymes<br />Agrivida: switchgrass, sorghum, corn<br />Ceres: non-food grasses<br />Edenspace Systems: corn, switchgrass<br />Farmacule BioIndustries: sugarcane, tobacco<br />Syngenta: corn<br />

27.
Strategies to Decrease or Modify Lignin Content in Energy Crops<br />

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Modification of Lignin Content<br />phenylalanine<br />lignin monomers<br />Source: Simmons et al. (2010) Curr. Opin. Plant Biol. 13:313-320<br />

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Strategies for Modification of Lignin Content<br />Down-regulate coordinated expression of lignin biosynthetic genes.<br />Knock out genes encoding enzymes that catalyze synthesis of lignin monomers. <br />Modify monomers or change relative composition of monomers to create more readily-degraded polymers.<br />Block or down-regulate polymerization of monomers.<br />Enhance lignin degradation, e.g. by overexpressing degradative enzymes.<br />Need to avoid deleterious effects on plants caused by low lignin concentrations.<br />

30.
Modification of Lignin Content<br />Downregulation of caffeic acid 3-O-methyltransferase (COMT) in switchgrass led to 12% reduction in lignin and 33% increase in ethanol production.<br />

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Modification of Lignin Content<br />Creation of new enzymes to methylate lignin monomers at the highlighted OH groups to prevent polymerization.<br />

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Companies Pursuing Modified Lignin Content<br />ArborGen: purpose-growntrees with altered lignin biosynthesis<br />

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Uses of Genomics to Enhance Energy Crops<br />

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Completed Energy Crop Genome Sequences<br />Corn<br />Soybean<br />Sorghum<br />Rice<br />Cottonwood<br />Jatropha<br />B. distachyon(model grass)<br />

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Impact of Biotechnology Regulations on Biofuels<br />

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US Regulation of Transgenic Plants<br />USDA regulations require agency notification for field tests of most transgenic plants.<br />Permits generally required for industrial applications (submission 120 days in advance).<br />Commercial approvals via “deregulation” petitions.<br />Tens of thousands of field tests and 81 commercial approvals under these rules (Syngenta corn: first transgenic energy crop to win commercial approval). <br />Approvals can take 1-4 years and may require Environmental Impact Statements.<br />

37.
Canadian Regulation of Transgenic Plants<br />Food Inspection Agency regulations govern field testing (“confined release”) and commercialization (“unconfined release”) of plants with novel traits (“PNTs”).<br />Regulations similar to US regulations.<br />Many field tests and commercial approvals have been authorized under these regulations.<br />

38.
Field Uses of Transgenic Plants for Biofuels: US and Canada<br />Infinite Enzymes, Edenspace, Targeted Growth, ArborGen, Ceres: transgenicfield trials in the U.S. of various species, including tobacco, corn, Eucalyptus, switchgrass, Miscanthus, 2006-present.<br />Agrisoma, Targeted Growth: field trials in Canada of engineered Brassica, soybean, Camelina, with improved traits for biofuels, such as oil quality, content and seed size, 2009-present.<br />Numerous academic groups: field trials in both countries of transgenic energy crops, model species.<br />

39.
European Regulation of Transgenic Plants<br />Directive 2001/18/EC covers experimental release of GMOs into the environment, and the placing on the market of GMOs.<br />R&D field tests are reviewed and approved by the competent authority of the EU member in which the experimental release is to take place.<br />Commercial approvals are made by national authority, subject to possible objections by other member states.<br />This program has been controversial, particularly with regard to food uses of GMO plants.<br />

40.
Field Uses of Transgenic Plants for Biofuels: Europe<br />Biogemma, modified hypolignified tall fescue, corn modified for lignin biosynthesis pathway, France 2003.<br />INRA, modified poplars for wood properties and bioenergy production, France 2007.<br />Idén Biotechnology, maize lignification for the improvement of digestibility and bioethanol production, Spain 2010. <br />ABBA Gaia, Nicotiana glauca modified as an energy crop, Spain 2010.<br />Warsaw University of Life Sciences, modified poplar, Poland, 2010.<br />Public University of Navarre, maize modified for starch, sugar content, Spain, 2010.<br />Date of proposal is shown. Status of all field tests is not known.<br />

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Commercialization Status and Prospects<br />

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Prospects for the Future: Commercialization Challenges<br />Use of transgenic energy crops will face challenges:<br />Will developer’s profit be sufficient to recoup R&D costs?<br />Crop segregation needed to preserve value for grower.<br />Competition from conventionally-bred varieties.<br />Availability of sufficient land to grow crops.<br />Regulatory hurdles in some countries.<br />For certain crops, need to avoid contamination of food supplies.<br />

43.
Thank you very much<br />David J. Glass, Ph.D.<br />D. Glass Associates, Inc.<br />124 Bird Street<br />Needham, MA 02492 USA<br />Phone +1 617-653-9945<br />DGlassAssc@aol.com<br />www.dglassassociates.com<br />More detailed information on the subjects presented in this talk can be found at my blog:<br />http://dglassassociates.wordpress.com<br />