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
12. Enhancing Carbohydrate, Polysaccharide Content

Increase concentration of glucose, other sugars

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.
25. Expression of Degradative Enzymes
Edenspace Systems: Express cellulases to enhance cellulose breakdown

Agrivida: Timed, inducible expression of cell-wall degrading enzymes

Agrivida, Inc. 2011
dormant enzyme
crop harvest and
enzyme activation
activated enzyme
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 acid3-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 coversexperimental 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.
Idn 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 developers 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