University of California, Merced igem 2012

Transcription

1 H2 E. hydro Express Streamlining Bacterial Production of Hydrogen Gas H2 H2 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. García-Ojeda 1 1. School of Natural Science, University of California, Merced 2. School of Engineering, University of California, Merced

2 Foundation 2012

3 Background Global Need for Energy Fossil fuel resources Finite, non-renewable Harmful carbon byproducts Other biofuels? flickr user aero nerd. Veziroglu TN and Sahin S. (2008) 21st Century s energy: Hydrogen energy system. Energy Conversion and Management 49 :

4 Background Hydrogen as Renewable Energy Source Benefits Efficient: fuel + energy carrier Reduces dependency on petroleum Produced domestically 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 : Jensen J et al. (2011) Hydrogen Implementing Agreement: Hydrogen. International Energy Agency, IEA CERT Workshop: Spormann AM et al. (2005) Metabolic Engineering of Hydrogen Production in Cyanobacterial Heterocysts. Stanford: GCEP Technical Report: 1-2.

5 Project Overview H2 H2 Biohydrogen Gas Production via Fermentation Goal: Exploit fermentative capabilities in E. coli to produce H 2 Strategy: Knockouts & Insertions New Metabolic Pathway H 2 H 2 Lee D et al. (2011) Dark fermentation on hydrogen production: pure culture. Bioresource Technology 102: Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): Hallenbeck PC and Benemann JR. (2002) Biological hydrogen production; fundamentals and limiting processes. International Journal of Hydrogen Energy 27:

6 Design & Strategy Glycolysis Produces H + and NADH 2 NAD +, 2ADP, 2P i 2 NADH, 2 ATP, 2 H +

7 Design & Strategy Pyruvate decarboxylase 2 Pyruvate H + 2 CO NAD + Acetaldehyde dehydrogenase 2 NADH + 2 H + 2 Acetyl-CoA

8 Design & Strategy FO Replenishes NAD + and Contributes to H 2 Production NADH Ferredoxin ox H 2 Ferredoxin oxidoreductase FO gene missing in E. coli Hydrogenase NAD + Ferredoxin red H +

9 Design Project & Overview Strategy Overview of Fermentation in E. coli Mixed Acid Fermentation Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

10 Design & Strategy Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: Targeted Pathway for Modification

11 Design & Strategy Modifications to E. Coli Fermentation Pathway 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.

12 Design & Strategy Overview of Engineered H 2 Production Pathway Pyruvate H+ Formate Lactate 1 Acetyl-CoA 2 Acetaldehyde 2 NADH + 2 H + 2 NAD + H Ethanol

13 Insert Design Methylophilales bacterium Acetaldehyde dehydrogenase (AD) E. coli W Ferredoxin Oxidoreductase (FO) Gibson Assembly

14 Plasmid Insertion Plasmid Insertion PD H 2 H 2 H 2 H 2 E. coli FMJ39 H 2 Insert ldha PD pflb FO adhe AD (mhpf) Knockout ldha pflb

15 Results PCR Cloning of Parts

16 Results Successful Transformation of FMJ39

17 Next Steps Transduction to knock out last gene (adhe) Ensure correct inserted sequences

18 Next Steps Testing for Hydrogen Gas Source: Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): Setup to measure the hydrogen gas

19 Future Directions Future Projects & Applications Cellulose Glucose

20 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

21

22 Thank You!