A New Biorefinery Platform Intermediate Principal Investigator: Doug Cameron Project Managers: Jim Millis, Paris Tsobanakis 2005 OBP Bi-Annual Peer Review Integrated Biorefinery Platform November 16, 2005

Overview Scope of Work Plan Cargill, in partnership with Codexis, is developing a fermentation route to a novel platform chemical, 3-hydroxypropionic acid (3-HP) We are also collaborating with PNNL to develop novel catalytic process for the conversion of 3-HP to acrylic acid and acrylamide Glucose3-HP Acrylic Acid Acrylamide Microbial fermentation Chemical catalysis

Overview Award was for $6.09 million in matching DOE funds for a 3 year program (total $12.2 MM), representing a 50/50 cost share Biotech/Codexis work started in May 2003; Chemical conversion/ PNNL work started in December, 2003 The pace of the program was slowed after one year as commercial activities lagged technology development. ‒ $0.60 MM received in FY03 (May to September) ‒ $1.66 MM received in FY04 ‒ $0.77 MM received in FY05 ‒ Request for FY06 and beyond to be submitted by end of November ‒ 50% complete ‒ Completion of the program will require a one year extension

Overview The program is currently in Development Phase Barriers addressed 3-HP is a novel platform chemical (identified as a Top Ten product) with the potential to impact both large, commodity markets and new, emerging markets Technical barriers addressed: ‒ Specific improvements in metabolic pathway engineering to yield highly productive, high yield fermentation technology ‒ Novel product recovery and process integration ‒ Design of new catalysts and processes (for derivatives of 3-HP)

Project Goals and Objectives Cargill, through collaboration with their partners Codexis and PNNL will develop a new bio-based platform technology to produce a portfolio of products based on 3-HP produced by the fermentation of carbohydrates. This project will deliver an organism and process for 3-HP production, and catalysts and complete process concepts suitable for piloting and scale-up for industrial production. Our current timeline fits well with the FY08 Solicitation schedule

3-hydroxypropionic acid 1,3-propanediol EEP acrylamide acrylic acid malonic acid poly(hydroxypropionate) Project Strategic Fit

1994 = 100 Project Strategic Fit Market dynamics have emerged to make 3-HP conversion to acrylic as an attractive alternative to propylene oxidation

PropylenePolypropylene Acrylonitrile Oxo Chemicals (butanol, 2-EH) Propylene oxide Cumene Isopropyl alcohol Polygas chemicals (nonene, dodecene) Other chemical uses Polyurethane polyols Propylene glycols Glycol ethers Adiponitrile ABS/SAN resins Acrylamide Nitrile rubber Phenol Acetone Acrylic acid 57% Despite a global market of 6.6 billion pounds for acrylic acid, propylene used for acrylic production is a small portion of propylene supply, with price driven principally by polypropylene Growth requires one new world class plant every year 10% 4% 12% 7% 8% 4% 3% Project Strategic Fit

Sponsoring BU within Cargill is Industrial Bioproducts, a member of the Emerging Business Platform This project enables a lower cost (and less price volatile) route to acrylic acid from renewable sugars. Joint analysis of production economics with an industry leader indicates a potential manufacturing cost advantage vs. propylene oxidation of >5¢ per pound for a Midwest plant. Energy consumption would be reduced 61% compared to the petrochemical route. By 2020, the acrylic acid product alone would displace 7,200 barrels of oil/day with domestically produced corn. The biorefinery route to acrylic acid would eliminate 600,000 lbs of SO x, 1 million lbs of NO x and 2.8 billion lbs of CO 2 annually. Project Strategic Fit

Project Approach L-  -alanine Pyruvate Glucose  -alanine 3-HP malonate semialdehyde – Pyruvate / alanine aminotransferase 2 – Alanine 2,3-aminomutase 3 –  -alanine aminotransferase 4 – 3-HP dehydrogenase α-ketoglutarate glutamate glutamate α-ketoglutarate NADH ATP

Project Collaboration Glucose3HP Acrylic Acid Acrylamide Microbial catalysis/ fermentation Chemical catalysis PNNL: Catalyst & process development Codexis: Enzyme & pathway evolution Cargill: Host strain construction & fermentation process Cargill: Process design, integration & economics Task Group 1Task Group 2Task Group 3

Project Tasks Task Groups 1.Biochemical pathway optimization 2.Host strain construction and fermentation process development 3.Catalyst screening and development, process development and process integration for the conversion of 3-HP to acrylic acid

Project Tasks Task Group 1: Biochemical pathway optimization (Codexis) Targets Improve performance of Enzyme 2 to meet productivity targets Balance supporting pathway enzymes to maximize productivity and yield Milestones Performance measured by fermentation productivity (see Task Group 2) Technical Risks Enzyme 2 does not meet activity target Future plans Continue optimization of Enzyme 2 Perform pathway shuffling to optimize pathway performance Random Phylogenetic – semisynthetic Targeted Hit shuffling ProSAR WT Parent Rd1 Rd2 Rd3 Rd4 Rd5 Rd6Rd7 Rd8 Rounds of shuffling Relative improvement

Project Tasks Task Group 2: Host strain construction and fermentation process development (Cargill) Targets Host strain selected and pathway engineered into strain Pathway constructed and 3-HP produced Fermentation optimization to meet economic targets Milestones Specific productivity targets communicated to DOE (confidential) Technical Risks Targets for high productivity and titer are not met Future plans Incorporate improved variants of Enzyme 2 into pathway Improve yield and productivity of fermentation process

Project Tasks Task Group 3: Catalyst screening and development, process development and process integration for the conversion of 3-HP to acrylic acid (PNNL, Cargill) Targets Catalysts screened and selected for the dehydration of 3-HP to acrylic acid Catalyst & process conditions demonstrated near quantitative conversion and selectivity Milestones Milestones met for pure 3-HP Technical Risks Catalyst robustness/ effect of impurities Future plans Demonstrate extended catalyst life Demonstrate catalyst performance on 3-HP produced by fermentation Process integration

This project enables the first biobased route to acrylic acid, a commodity product with a global market approaching 7 billion pounds per year. Market & Customers Meeting economic targets for acrylic will also enable other 3-HP derivatives. –1,3-propanediol –malonic acid and others

Cargill’s route to acrylic acid through 3-HP is advantaged over other technology options –Propane oxidation is unproven and does not meet DOE OBP objectives –Lactic acid dehydration pursued by many parties without success –Future may allow use of biomass feedstock for Cargill process, thus driving costs downward Relative attractiveness of 3-HP to acrylic technology will depend on propylene price, but current forecast for high prices and volatility provides the basis for optimism Competitive Advantage

Progress and Accomplishments Production of 3-HP from glucose demonstrated by a novel pathway Catalyst discovered for 3-HP to acrylic acid process Dehydration reactor designed to overcome equilibrium limitations

Future Work Development Stage Plans Due to a reduced rate of spend in FY04 and FY05, Cargill requests an extension of one year to complete the program. New proposed date for completion is May ‘07. Plans are to accelerate the spend over the next 18 months. Interim milestones and commercial plans communicated to DOE in July 2005 review (confidential). Our current timeline fits well with the FY08 Solicitation schedule