streamlining bacterial production of hydrogen...
TRANSCRIPT
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/
Ba
ckg
rou
nd
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/
Ba
ckg
rou
nd
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
Futu
re D
ire
ctio
ns
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!