This is a presentation I made on February 2, 2010 at the EUEC 2010 conference in Phoenix. The talk included an overview of the approaches being pursued to use biotechnology to improve microorganisms, algae and plants for biofuel production and the companies pursuing these strategies, and discussion of the impact of biotech regulations on these projects and the prospects for use of engineered organisms in commercial biofuel production. You can find more detailed information on the topics discussed in this talk on my blog at http://dglassassociates.wordpress.com.

1. Prospects for the Commercial Use of Genetic Engineering in Biofuel Production David J. Glass, Ph.D. D. Glass Associates, Inc. Presented at EUEC 2010, Phoenix, AZ February 2, 2010

2. Overview: Prospects for Use of Advanced Biotechnology for Biofuel Production Summary of technology strategies. Companies applying advanced biotechnology to biofuels. Impact of biotechnology regulations on biofuels. Commercialization status and prospects.

3. Technology Strategies

4. Biotechnologies Applicable to Biofuels Classical microbial mutation and selection Plant breeding Recombinant DNA Directed evolution DNA shuffling Metabolic engineering Synthetic biology

6. Engineer microorganisms to manufacture novel or improved industrial enzymes.

8. Genetic Engineering Strategies: Plants Overexpress cell-wall hydrolysis enzymes. Cellulases, Hemicellulases, Ligninases. Increase plant biomass, cellulosic biomass. Cellulose biosynthetic enzymes. Lignin modification to reduce need for pretreatment. Down-regulate lignin biosynthesis. Regulated gene expression, so traits are active only when needed.

9. Companies applying advanced biotechnology to biofuels

10. Companies Using or Developing Modified Microorganisms: Ethanol BioEnergy International DuPont Danisco Glycos Biotechnologies GreenTech America Joule Biotechnologies Mascoma Microbiogen (non-GMO) Promethegen Qteros TMO Renewables Verenium Zymetis Note: Several of these companies are also developing other fuels.

11. Companies Using or Developing Modified Microorganisms: Biodiesel, Other Fuels Biodiesel, petroleum fuels Amyris Biotechnologies Codexis Glycos Biotechnologies Joule Biotechnologies LS9 Promethegen Sequesco Synthetic Genomics Butanol, Isobutanol Bioenergy International Butamax (DuPont/BP) Cobalt Biofuels Gevo Green Biologics, Ltd. METabolic Explorer TetraVitae Bioscience

12. Companies Using Newer Microbial Technologies Amyris Biotechnologies: synthetic biology Codexis: DNA shuffling Glycos Biotechnologies: metabolic engineering Joule Biotechnologies: synthetic biology LS9: synthetic biology Synthetic Genomics: synthetic biology TMO Renewables: thermophilic microorganisms Verdezyne: synthetic biology, metabolic engineering

13. Companies Manufacturing Enzymes for Biofuels using Modified Organisms Microorganisms AB Enzymes Danisco/Genencor Royal DSM N.V. Dyadic International Iogen Corporation Novozymes A/S Protéus Plants Infinite Enzymes (transgenic plants) Medicago (transient expression) Syngenta (transgenic plants)

14. Companies Reported to be Developing Genetically Modified Algae for Biofuels Algenol Biofuels Aurora Biofuels AXI LLC Global Green Solutions Sapphire Energy Solazyme Synthetic Genomics Targeted Growth Note: companies included based on press, Internet reports: use of GMOs not necessarily confirmed on company websites. Not all these companies are necessarily using genetic engineering.

15. Companies Developing Transgenic Plants for Biofuels Conventional Feedstocks Agrivida: corn ArborGen: purpose-growntrees Edenspace Systems: corn Targeted Growth: corn Note: some of these companiesare also developing newer feedstocks.

16. Companies Developing Transgenic Plants for Biofuels Newer Feedstocks Agragen: Camelina Agrisoma: BrassicaandJatropha Agrivida: switchgrass, sugarcane, sorghum, others Ceres: non-food grasses Edenspace Systems: switchgrass Evogene: canola, soybean, others

17. Companies Developing Transgenic Plants for Biofuels Newer Feedstocks Farmacule BioIndustries: sugarcane, tobacco Mendel Biotechnology: grasses, others Metabolix: switchgrass, oil crops, others SG Biofuels: Jatropha Targeted Growth: Camelina, canola, others

18. Impact of Biotechnology Regulations on Biofuels

19. Overview of U.S. Regulation of Environmental Biotechnology Environmental Protection Agency Microbial pesticides. Plant pesticides (e.g. transgenic plants). Engineered microorganisms not regulated by other agencies (e.g. nitrogen fixation, bioremediation, industrial applications). U.S. Department of Agriculture Transgenic plants, including plants producing industrial or pharmaceutical products. Transgenic animals.

20. EPA Regulation of Engineered Microorganisms EPA regulations under the Toxic Substances Control Act (TSCA) cover industrial uses of microorganisms not regulated by other agencies. Applies to “intergeneric” organisms for industrial enzyme production, biotreatment, other bioprocessing uses. Applicability to algae not certain. Submit Microbial Commercial Activity Notice (MCAN) with data package 90 days before starting commercial use. Most research and pilot projects are not regulated. Some exemptions available for commercial use.

21. USDA Regulation of Transgenic Plants Regulations issued in 1987 cover outdoor uses of most transgenic plants. Rules have been continually relaxed, most agricultural uses require only agency notification. Starting in 2003, permits now required for industrial, pharmaceutical, phytoremediation applications. Permit applications: must be submitted 120 days in advance of proposed field use. Commercialization via “delisting” petition process. USDA currently considering revisions to regulations.

22. Regulation of Engineered Algae Jurisdiction of EPA, USDA under biotech regulations unclear. Proposed use of engineered algae in pharmaceutical production in Hawaii in 2005 generated controversy, no federal agency claimed jurisdiction: state law and state courts ultimately determined outcome. 2008 USDA opinion letter to Coastal BioMarine stated no USDA oversight over engineered algal strain if no “plant pest” sequences, and no oversight for smaller-scale use in contained reactors. Reactor design may be critical in determining regulatory status.

23. Impact of Biotechnology Regulations Would not necessarily affect all biofuel organisms, exemptions may be available. For those projects affected, some additional activities and expense may be required. Straightforward path to commercial approval, with many prior successes in agricultural, industrial biotechnology. The regulatory process can be successfully managed.

24. Commercialization Status and Prospects

25. Commercial Status of Modified Microorganisms in Biofuels Verenium: only MCAN reviewed by EPA under TSCA for modified ethanol production strain. Demonstration or pilot plants in operation or under construction: Ethanol production: BioEnergy, DuPont Danisco, Iogen, Mascoma, Qteros, Verenium. Gevo: demonstration butanol plant. Amyris: pilot plant for petroleum-equivalent fuels. Pilot or commercial algae plants: Algenol, Aurora, Sapphire, Solazyme, Solix (not necessarily using engineered algae).

26. Field Uses of Transgenic Plants for Biofuels Syngenta: Approval for commercial use of amylase-expressing corn in several countries, approval pending in U.S. since 2005. Agrisoma: field trials in Canada of engineered Brassica and soybean, improved in oil quality, content and seed size for biodiesel use, 2009. Infinite Enzymes, Edenspace, Targeted Growth, ArborGen: field trials in the U.S. of various species, including tobacco, corn, Eucalyptus, expressing biodegradative enzymes, 2006-09.

27. Notable Business Deals Involving Advanced Biotechnology for Biofuels BP/Martek: Conversion of sugars to biodiesel. BP/Verenium: Cellulosic biofuel development. Chevron/Solazyme: Biodiesel production from algae. Dow Chemical/Algenol: Pilot plant for algal production of ethanol. ExxonMobil/Synthetic Genomics: Synthetic algal strains for biofuel production. Royal Dutch Shell/Codexis: Novel methods for conversion of non-food biomass to biofuels. Royal Dutch Shell/HR Petroleum: marine algae for biofuels.

28. Prospects for the Future: Commercialization Challenges Use of modified organisms will face the same challenges as other biofuel applications: Scale-up to commercial scale. Process economics. Competition from other biological or physical methods of biofuel manufacture. Feedstock availability and cost; conflict with food use of crops Availability of sufficient land to grow feedstocks or to build reactors.

29. Prospects for the Future: Commercialization Challenges But use of modified organisms will also face unique challenges: Technical hurdles to accomplish performance goals. Recovering the high R&D costs of advanced biotech. Government policies, including biotechnology regulation, especially outside the U.S. Public acceptance of biotechnology solutions.

30. Prospects for the Future: Commercialization Strategies Integration of biological processes with other downstream process improvements. Diversification: Augment energy production by co-production of value-added end-products: specialty chemicals, bioplastics. Entry of tree, plant genetics companies into biofuels. Carbon capture: design systems to capture CO2 streams for use as biofuel feedstock. Novel feedstocks: use of cellulosic waste, novel nonfood crop species as biofuel feedstocks.

31. Thank you very much David J. Glass, Ph.D. D. Glass Associates, Inc. 124 Bird Street Needham, MA 02492 Phone 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