University of California, Merced iGEM 2012

Paul Barghouth2, John Flicker1, Israel Juarez-Contreras2, Marwin Ko2, Norman Luong2, Rumman Razzak1, Sunny Seth1, Michael Urner1,2, Duc Vo1, Catherine

Vu1, Yale Yuen2, Nosherwan Zahid1, Marcos E. García-Ojeda1 1. School of Natural Science, University of California, Merced

2. School of Engineering, University of California, Merced

E. hydro Express Streamlining Bacterial

Production of Hydrogen Gas

H2 H2

H2

Foundation

2012

Global Need for Energy

• Fossil fuel resources

– Finite, non-renewable

– Harmful carbon byproducts

Other biofuels?

http://www.homepagedaily.com/Pages/article10410-dwindling-fossil-fuels-and-our-food-system.aspx

http://youevolving.wordpress.com/tag/fossil-fuels/

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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

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

http://www.hydrogen.co.uk/

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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.

• Goal:

Exploit fermentative capabilities in E. coli to produce H2

• Strategy:

Knockouts &

Insertions

New Metabolic Pathway

H2

Project Overview

Biohydrogen Gas Production via Fermentation

H2 H2

H2

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.

2 NAD+, 2ADP, 2Pi

2 NADH, 2 ATP, 2 H+

De

sig

n &

Str

ate

gy

Glycolysis Produces H+

and NADH

2 Pyruvate

H+

2 CO2 +

Pyruvate decarboxylase

2 NAD+

2 NADH + 2 H+

2 Acetyl-CoA

Acetaldehyde dehydrogenase

De

sig

n &

Str

ate

gy

FO Replenishes NAD+ and Contributes to H2 Production

NADH

NAD+

Ferredoxinox

Ferredoxinred

H2

H+

Hydrogenase Ferredoxin oxidoreductase

De

sig

n &

Str

ate

gy

FO gene missing in

E. coli

Overview of Fermentation in E. coli

Pro

ject

Ov

erv

iew

Mixed Acid Fermentation

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

De

sig

n &

Str

ate

gy

De

sig

n &

Str

ate

gy

Targeted Pathway for Modification

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

Modifications to E. Coli Fermentation Pathway

De

sig

n &

Str

ate

gy

Mixed Acid Fermentation

+ Acetaldehyde dehydrogenase

+ Pyruvate Decarboxylase

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

Pyruvate

Acetyl-CoA

Acetaldehyde

Ethanol

Formate

Lactate

1

2

H+

2 NAD+ 2 NADH + 2 H+

3 H2 4

De

sig

n &

Str

ate

gy

Overview of Engineered H2

Production Pathway

E. coli W Methylophilales bacterium

Acetaldehyde dehydrogenase (AD) Ferredoxin Oxidoreductase (FO)

Gibson Assembly

Insert Design

E. coli FMJ39

ldhA

pflB

adhE

Insert Knockout

PD ldhA

FO pflB

AD (mhpF)

H2

Plasmid Insertion

H2

H2

H2

H2

Plasmid Insertion

PD

PCR Cloning of Parts R

esu

lts

Successful Transformation

of FMJ39

Re

sult

s

Next Steps

• Transduction to knock out last gene (adhE)

• Ensure correct inserted sequences

Testing for Hydrogen Gas

Setup to measure the hydrogen gas

Ne

xt

Ste

ps

Source: Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): 30-39.

Future Projects & Applications

Cellulose Glucose

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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!