Presentation by David Glass at 2011 World Biofuels Markets Conference, Rotterdam, the Netherlands, March 22, 2011. This presentation describes approaches being taken to use advanced biotechnology to improve the plant species being used as biofuel feedstocks. This was part of the "Energy Crops" sessions at the conference, which Dr. Glass moderated.

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

2. Overview of Technology Strategies

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

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

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

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

7. Strategies to Enhance Lipid Content in Energy Crops

8. Enhancing Lipid Content Enhance fatty acid biosynthesis Overexpress ACCase Enhance triacylglycerol synthesis Overexpress DGAT, other pathway enzymes

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

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

11. Strategies to Enhance Polysaccharide Content in Energy Crops

13. Hexose, sucrose transporters

14. Glucose signaling

15. Increase cellulose, hemicellulose content in cell walls

16. Cellulose synthase

17. Glucosyltransferase

18. Enhance starch biosynthesis

19. Starch synthases

20. Branching, debranching enzymesSource: Sticklen (2008) Nature Reviews Genetics 9:433-443

21. Companies Pursuing Enhanced Polysaccharide Content Chromatin: switchgrass, Miscanthus, sorghum FuturaGene: switchgrass, Miscanthus, hybrid poplar and willow Mendel Biotechnology:grasses, others Targeted Growth: corn

22. Strategies to Express Degradative Enzymes in Energy Crops

23. Expression of Degradative Enzymes Express thermostable amylase in plant tissue, to enhance starch breakdown.

24. Expression of Degradative Enzymes Express enzymes to enhance breakdown of hemicellulose.

26. Companies Expressing Degradative Enzymes Agrivida: switchgrass, sorghum, corn Ceres: non-food grasses Edenspace Systems: corn, switchgrass Farmacule BioIndustries: sugarcane, tobacco Syngenta: corn

27. Strategies to Decrease or Modify Lignin Content in Energy Crops

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

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

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

31. Modification of Lignin Content Creation of new enzymes to methylate lignin monomers at the highlighted OH groups to prevent polymerization.

32. Companies Pursuing Modified Lignin Content ArborGen: purpose-growntrees with altered lignin biosynthesis

33. Uses of Genomics to Enhance Energy Crops

34. Completed Energy Crop Genome Sequences Corn Soybean Sorghum Rice Cottonwood Jatropha B. distachyon(model grass)

35. Impact of Biotechnology Regulations on Biofuels

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

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

38. Field Uses of Transgenic Plants for Biofuels: US and Canada Infinite Enzymes, Edenspace, Targeted Growth, ArborGen, Ceres: transgenicfield trials in the U.S. of various species, including tobacco, corn, Eucalyptus, switchgrass, Miscanthus, 2006-present. 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. Numerous academic groups: field trials in both countries of transgenic energy crops, model species.

39. European Regulation of Transgenic Plants Directive 2001/18/EC covers experimental release of GMOs into the environment, and the placing on the market of GMOs. 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. Commercial approvals are made by national authority, subject to possible objections by other member states. This program has been controversial, particularly with regard to food uses of GMO plants.

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

41. Commercialization Status and Prospects

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

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