E. hydro Express Streamlining Bacterial Production of Hydrogen Gas H 2 H 2 H 2 University of California, Merced iGEM 2012 Paul Barghouth 2 , John Flicker 1 , Israel Juarez-Contreras 2 , Marwin Ko 2 , Norman Luong 2 , Rumman Razzak 1 , Sunny Seth 1 , Michael Urner 1,2 , Duc Vo 1 , Catherine Vu 1 , Yale Yuen 2 , Nosherwan Zahid 1 , Marcos E. Garc í a-Ojeda 1 1. School of Natural Science, University of California, Merced 2. School of Engineering, University of California, Merced
Foundation 2012
Global Need Background for Energy • Fossil fuel resources – Finite, non-renewable http://youevolving.wordpress.com/tag/fossil-fuels/ – Harmful carbon Other biofuels? byproducts http://www.homepagedaily.com/Pages/article10410-dwindling-fossil-fuels-and-our-food-system.aspx flickr user aero nerd. http://www.motherjones.com/blue-marble/2009/06/climate-bill-biofuel-boondoggle Veziroglu TN and Sahin S. (2008) 21st Century’s energy: Hydrogen energy system. Energy Conversion and Management 49 : 1820– 1831 .
Hydrogen as Renewable Energy Source Background Benefits – Efficient: fuel + energy carrier – Reduces dependency on petroleum – Produced domestically http://www.hydrogen.co.uk/ – Clean energy Challenges • High fuel cost, low availability • Most hydrogen gas produced by thermochemical reformation of fossil fuels still emit greenhouse gases Veziroglu TN and Sahin S. (2008) 21st Century’s energy: Hydrogen energy system. Energy Conversion and Management 49 : 1820– 1831. Jensen J et al. (2011) Hydrogen Implementing Agreement: Hydrogen. International Energy Agency, IEA CERT Workshop: 1-23. Spormann AM et al. (2005) Metabolic Engineering of Hydrogen Production in Cyanobacterial Heterocysts. Stanford: GCEP Technical Report: 1-2.
Project Overview H 2 H 2 H 2 Biohydrogen Gas Production via Fermentation • Goal: Exploit fermentative capabilities in E. coli to produce H 2 • Strategy: Knockouts New H 2 & Metabolic Insertions Pathway Lee D et al. (2011) Dark fermentation on hydrogen production: pure culture. Bioresource Technology 102: 8393-8402. Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): 30-39. Hallenbeck PC and Benemann JR. (2002) Biological hydrogen production; fundamentals and limiting processes. International Journal of Hydrogen Energy 27: 1185-1193.
Glycolysis Produces H + and NADH Design & Strategy 2 NAD + , 2ADP, 2P i 2 NADH , 2 ATP , 2 H +
Design & Strategy 2 Pyruvate Pyruvate decarboxylase H + 2 CO 2 + 2 NAD + Acetaldehyde dehydrogenase 2 NADH + 2 H + 2 Acetyl-CoA
FO Replenishes NAD + and Contributes to H 2 Production Design & Strategy Ferredoxin ox NADH H 2 FO gene Ferredoxin Hydrogenase missing in oxidoreductase E. coli NAD + H + Ferredoxin red
Overview of Fermentation in E. coli Design & Strategy Project Overview Mixed Acid Fermentation Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.
Targeted Pathway for Modification Design & Strategy Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.
Modifications to E. Coli Fermentation Pathway Design & Strategy Mixed Acid Fermentation + Pyruvate Decarboxylase + Acetaldehyde dehydrogenase Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.
Overview of Engineered H 2 Production Pathway Design & Strategy Lactate Pyruvate H+ Formate 1 Acetyl-CoA 2 Acetaldehyde 2 NADH + 2 H + 2 NAD + H 2 4 3 Ethanol
Insert Design Methylophilales bacterium E. coli W Acetaldehyde dehydrogenase (AD) Ferredoxin Oxidoreductase (FO) Gibson Assembly
Plasmid Insertion Plasmid Insertion PD H 2 H 2 H 2 H 2 Insert Knockout ldhA PD ldhA pflB H 2 FO pflB adhE AD (mhpF) E. coli FMJ39
PCR Cloning of Parts Results
Successful Transformation of FMJ 39 Results
Next Steps • Transduction to knock out last gene ( adhE ) • Ensure correct inserted sequences
Testing for Hydrogen Gas Next Steps Source: Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): 30-39. Setup to measure the hydrogen gas
Future Projects & Future Directions Applications Cellulose Glucose http://commons.wikimedia.org/wiki/File:Sunlight_Through_Leaves.jpg
Acknowledgements • School of Natural Sciences & School of Engineering, University of California, Merced • ASUCM • Dr. Giovannoni, Oregon State University • University of California, Davis • Yale University • Advisors: – Dr. Marcos E. Garcia-Ojeda – Dr. Wei-Chun Chin
Thank You!
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