Helioculture. Photobiocatalysis for Fuels and Chemicals Joule. Rights Reserved.

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2 Helioculture Photobiocatalysis for Fuels and Chemicals 2015 Joule. Rights Reserved.

3 About Joule Founded In 2007: Flagship Venture Labs Main office in Bedford, MA, USA Demonstration facility in Hobbs, NM, USA 65 Patents, Validations, and Allowances; 90+ Patent Applications 124 Employees, Including 27 PhDs Highly Experienced Management Team 2015 Joule. Rights Reserved. 3

4 Aligned With Expert Recommendations To Optimize Productivity EXPERT RECOMMENDATION ENGINEERED ORGANISM BIOCATALYTIC PRODUCTION MINIMIZE RESPIRATION MAXIMIZE PHOTOSYNTHETIC EFFICIENCY CONTROL BIOBURDEN Sheehan, et al. (1998) A look back at the US Department of Energy s aquatic species program. US DOE Closeout report TP National Algal Biofuels Technology Roadmap. (2009) HELIOCULTURE TECHNOLOGY Engineered pathways for synthesis of drop-in fuels: Ethanol or C 11 n- alkane Carbon switch to control carbon flux from CO 2 to either biomass or to fuel pathway 95% CO 2 conversion to fuel Continuous product synthesis and secretion; 8-12 week process cycle times Operation at high CO 2 to minimize carbon and energy loss Prevents carbon & energy diversion by (photo)respiration Directed evolution generates strains with improved photosynthetic efficiency (PE) PBR design optimizes light, gas & thermal management Process design minimizes contaminant penetration Cleaning regime for axenic operation in situ bioburden control solution Rosenberg, et al. (2008) A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution Curr Opin Biotechnol 19:430: 2015 Joule. Rights Reserved. Wijffels and Barbosa (2010) An outlook on microalgal biofuels. Science 329:796

5 Photobiocatalyst Chassis: Cyanobacterium Synechococcus! Unicellular! Straightforward genetics! Rapid doubling! Concentrates CO 2! Euryhaline: Brackish, sea, fresh and produced water! Thermotolerant species for thermal management! Produces continuously for weeks in closed PBR! No biofilm or fouling 2015 Joule. Rights Reserved. 5

6 Fuel Synthetic Pathways CO 2 CO 2 PHOTOSYNTHE SIS NADPH NADP + Pyruvate decarboxylase Acetaldehyde Alcohol dehydrogenase Ethanol Pyruvate OR PHOTOSYNTHE SIS PHOTOSYNTHE SIS NADPH NADP + 4 Fdx red 4 Fdx ox O 2 H 2 O Acyl-ACP CENTRAL METABOLISM Acyl-ACP Reductase Alkanal Alkanal Deformylase (n-1) Alkane + Formate 2015 Joule. Rights Reserved. 6

7 Synthetic Switch for Carbon Partition Control Genes coding for enzymes that perform the chemistry of ethanol or alkane synthesis A synthetic switch controls the path of carbon from fixed CO 2 to fuel (w/ 95% efficiency) Additional engineering further improves light and CO 2 management CO 2 Carbo n Switch OFF Biomass Pyruvate Fuel SWITCH ON ETHAN OL CO 2 Switch ON Biomass BIOMAS S Pyruvate Carbo n Ethanol Production Rate KJ/ L / h Fuel Energy conversion rate KJ/ L / h % C-partition efficiency 2015 Joule. Rights Reserved. 7

8 Photobiocatalyst: Engineered for Continuous Fuel Synthesis CO 2 CO 2 Fixation Central Metabolism G3P BIOMASS Pyruvate Acetyl-CoA Pyruvate Decarboxylase ETHANO L SWITCH ALKANE SWITCH Engineered Pathways Acetaldehyde Alcohol Dehydrogenase Chromosome Ethanol SECRETIO N Ethanol Fatty acyl ACP Decarboxylase C 12 Aldehyde Alkenal Deformylase C 11 Alkane SECRETI ON C 11 n- Alkane Both ethanol and C 11 n-alkane can be blended directly into conventional fuels Ethanol/gasoline blends meet ASTM and DIN fuel standards C 11 n-alkane meets ASTM diesel standard at up to 50% blend; JetA, 25% blend 2015 Joule. Rights Reserved.

9 Photosynthetic Energy Conversion Efficiency (PE): Open vs Closed System Radiation in units of MJ/m 2 /year Baseline: Phoenix, AZ yearly average Energy in, MJ % Loss Energy Remaining, MJ Unfavorabl e Expenditur e Closed Process Advantage OPEN BATCH SOLAR ENERGY E out /E input % PE gal/acre/ yr CLOSED HELIOCULTURE E out / E input % PE gal/acre/yr TOTAL 110/ / k Ethanol 5k 15k C Biodiesel 11 PAR 110/ / Diesel 1. Weyer, Bush, Darzins, Wilson (2009) Theoretical maximum algal oil production. Bioenergy Res. 3: Robertson et al. (2011) A New Dawn for Industrial Photosynthesis. Photosyn Res 107: Joule. Rights Reserved. 9

10 PE Depends on Duration of Exposures to Light and Dark in PBR ATP/NADPH GENERATION RATE 100µsec ATP/NADPH UTILIZATION RATE 100msec NADPH PBR Light/Dark Regime CRITICAL VARIABLES Ratio of photic/dark depth Cell density Mixing rate 2015 Joule. Rights Reserved. 10

11 Effect of Light/Dark Cycling Frequency on PE- PAR PE-PAR 16% 14% 12% 10% 8% 6% 4% 2% 0% % ETHANOL PE-PAR Flash intensity = 2000 µe/m 2 / sec (Phoenix noon) Flash duration: 100µsec 25msec 200 Max PE- PAR Dark time 3.9msec 975msec % Joule. Rights Reserved. 9.8% 8.3% 7.7% % 6.4% 5.4% Frequency Solar Noon (2000 µe m -2 s -1 ) The frequency with which a cell visits the irradiated and dark zones of a photobioreactor has a direct effect on PE 1.8% 1.7% 0 Efficient Photosynthesis Photoinhibition Photooxidation Photobleaching ETHANOL The optimum frequency reflects matching of the rates of photon to ATP/NADPH (µsec), and CO 2 to fuel (msec) processes and elimination of energy losses photoinhibition Can be simulated with flashing light, varying flash intensity and duration, and the duration of the dark period between flashes, e.g., duty cycle and frequency

12 Systems Approach to Enhancing Productivity/ Longevity PHOTOBIOCATALYST Oxygen radical tolerance Carbon rerouting Metabolic regulation Reducing equivalents Light management O2 tolerance Ethanol/Alkane tolerance Thermal tolerance GENE 1 GENE 3 PROCESS Mixing Light management Length & depth Gas management Media components ph Thermal management Dissolved O2 & CO2 MUTAGENES IS CLONING GENE 2 REACTOR DESIGN >109 Clone s TRAIT SELECTI ON ~10 UpMutant s SCREENI NG LAB VALIDATION FIELD TESTING PROCESS & DESIGN Best Mutant CONSTRUCT COMPOSITE STRAIN Scale-Up 2015 Joule. Rights Reserved. 12

13 Exploring Genome Sequence Space For High PE Phenotypes Intuitive genetic engineering redirects carbon, controls gene regulation, resolves ROS Non-intuitive mutagenesis of genome and individual genes improves productivity >100 improved genotypes now being stacked into high productivity composites Cumulative Ethanol [mg/l] PRODUCTIVI T Y Paren t LONGEVITY Mutant Time [h] 2015 Joule. Rights Reserved. 13 Ethanol PE-PAR [%] 10% 8% 6% 4% 2% 0% Parent Time [h] 12,500g/a/y >700 hours Mutant

14 Bioburden Mitigation: Engineering Culture Competitiveness BIOBURDEN CONTROL Process contamination by foreign organisms can quickly lead to consumption of product Joule has engineered its biocatalyst for competitive advantage over contaminants Ethanol producing strains with the bioburden mitigation construct limit contaminant growth No ethanol is consumed BIOBURDEN PREVENTION Novel process plant design limits penetration points Pre-production cleaning and sterilization procedures eliminate contaminants EtOH (kj) 2015 Joule. Rights Reserved PARENT STRAIN No Added Contaminants BIOBURDEN MITIGATION STRAIN No Ethanol Consumption by Added Contaminants PARENT STRAIN Ethanol Consumption by Added Contaminants Time Repeated multi-week scaled outdoor experiments run axenically validate the strategy

15 Integrated Process Development Strain Development Strain Engineering Mutagenic evolution Light Management PBR Design Photon utilization Process Optimization Inoculum generation (OD, metabolic state) Induction for fuel (OD, inducer, timing) Operating ph and Buffer 2015 Joule. Rights Reserved. Thermal Management Proprietary low power technology Oxygen Management Mass transfer Strain tolerance Media additives 15 Product Recovery Strain tolerance Processing strip rate Media Chemistry Optimization Feeding of nutrients Additives

16 Helioculture: Biomass as Photobiocatalyst Harnesses solar energy and industrial waste CO 2 to produce fuels or chemicals Uses non-arable land; displaces no food sources; saline water Uses a genetically engineered photosynthetic cyanobacterium as a photobiocatalyst Genetic engineering introduces novel synthetic pathways and redesigns metabolic flux and regulation to achieve very high efficiency CO 2 conversion to product The photobiocatalyst directs the continuous conversion of solar energy to drive the synthesis and secretion of the desired product in week process cycles PBR design minimizes photon saturation, maximizes photosynthetic efficiency PBR is modular and scalable to industrial production 2015 Joule. Rights Reserved. 16

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