metabolic engineering of hydrogen production in...
TRANSCRIPT
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Metabolic Engineering of Hydrogen Production in Filamentous
Cyanobacteria
Alfred M. Spormann, Wing-On (Jacky) NgDepartments of Civil & Environmental Engineering, of Biological
Sciences, and of Geological & Environmental SciencesStanford University
September 19, 2006
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H2O
Photons (hPhotons (hνν ))PS II+PS I 2[H] + 1/2 O2
O2
PhysiologicalPath
H2
EngineeredPath
Hydrogen formation and oxygen evolution are naturally incompatible
Cell Mass
CO2
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H2O
Photons (hPhotons (hνν ))PS II+PS I 2[H] + 1/2 O2
O2
PhysiologicalPath
H2
EngineeredPath
Hydrogen formation and oxygen evolution are naturally incompatible
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Oxygen sensitivity of Hydrogenases
2H+
[e-] (reduced Ferredoxin)
H2O2
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http://www.wright.edu/biology/faculty/carmichael/labhome/green.jpg
The microorganisms of choice: Nitrogen-fixing Filamentous Cyanobacteria
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MH
PH
Fluorescence Images of Vegetativecells and Heterocysts
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http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG
Light Reaction in Heterocysts
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Function of heterocysts in filamentous cyanobacteria
O2
N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi
N2-ase reaction
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Engineering heterocysts forcyanobacterial hydrogen production
Engineered Anabaena
Anabaena wild type
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Plasmid for shydA expression
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H2 Chromatogram:
H2 Peak
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Relative transc ript levels o f nifD and shydA
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Relative transc ript levels o f nifD and shydA
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sHy dA protein leve l
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Integration of shydA into the nifHDKoperon in Anabaena 7120
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Hydrogen production from Anabaena7120 ex-conjugant
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Assembly and Maturation of Fe-only Hydrogenases
Alfred M. Spormann, Galit Meshulam-Simon, James R. Swartz
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6.8 kb6.8 kb
TheThe active site of Fe-only hydrogenase
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG Hypothetical hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG Hypothetical hydE hydFfdh
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Helper proteins involved in HydA maturation- Role of HydGEX -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydFfdh hydX
0
50
100
150
200
µ mol
H2 /
OD
∆hyaB ∆hyaB/∆hydG ∆hyaB/∆hydE ∆hyaB/∆hydX(hydA+)
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Acknowledgements• Jim Swartz• Chia-Wei Wang
• Peter Wolk (MSU)• Terry Thiel (UM, St.
Louis)
Funding:
Stanford SOE seed fund
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Fe-only Bi-directional Hydrogenases
2H+
[e-] (reduced Ferredoxin)
H2
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0
50
100
150
200
250
300
350
400
WT ∆hydA ∆hydA∆hydG ∆hyaB ∆hyaB∆hydG
µm
ol H
2 / O
D
Active HyaB
Active HydA
wt ∆hydA ∆hydA/∆hydG ∆hyaB ∆hyaB/∆hydG
Helper proteins involved in HydA maturation- Role of HydG -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydFfdh hydX
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0
50
100
150
200
250
300
350
400
WT ∆hydA ∆hydA∆hydE ∆hyaB ∆hyaB∆hydE
µm
ol H
2 / O
D
wt ∆hydA ∆hydA/∆hydE ∆hyaB ∆hyaB/∆hydE
Helper proteins involved in HydA maturation- Role of HydE -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydFfdh hydX
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0
50
100
150
200
250
300
350
400
WT ∆hydA ∆hydA∆hydX ∆hyaB ∆hyaB∆hydX
µm
ol H
2 / O
D
wt ∆hydA ∆hydA/∆hydX ∆hyaB ∆hyaB/∆hydX
Helper proteins involved in HydA maturation- Role of HydX -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydFfdh hydX
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0
50
100
150
200
250
300
350
400
WT ∆hydA ∆hydA∆hydE ∆hyaB ∆hyaB∆hydE
µm
ol H
2 / O
D
wt ∆hydA ∆hydA/∆hydE ∆hyaB ∆hyaB/∆hydE
Helper proteins involved in HydA maturation- Role of HydE -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG l hydE hydFfdh hydX
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0
50
100
150
200
250
300
350
400
WT ?hydA ?hydA?hydX ?hyaB ?hyaB?hydX
µmol
H2/ O
D
wt ∆hydA ∆hydA/∆hydX ∆hyaB ∆hyaB/∆hydX
Helper proteins involved in HydA maturation- Role of HydX -
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydF
SO3920 SO3922 SO3923 SO3924 SO3925 SO3926SO3921
hydALarge subunit
hydBSmall subunit
hydG hydE hydFfdh hydX
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Solar Energy
Microorganisms, Plants
O2CO 2H2O Biomass
Biological Ecosystems
Fossil Fuels
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Solar Energy
Sustainable clean energyFuel cell engines
H2O O2 H2
Engineered Microorganisms
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H2O
Photons (h ν )
PS II+PS I 2[H] + 1/2 O 2 O 2
PhysiologicalPath
Cell Mass
CO2
H2
EngineeredPath
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Filamentous Cyanobacteria
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Anabaena sp.
Heterocysts (Anaerobic, N2-Fixation)
Vegetative cells (Oxygenic Photosynthesis)
Source: http://web.uvic.ca/~h2o/Phyto2/Anabaena_affinis.jpg
Unique biological adaptation of filamentous cyanobacter
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www.chem.cmu.edu/.../ achim/research/magneto.html
Nitrogenase reactionO2
N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi
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Programmed Heterocyst Differentiation
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Indirect (2-Stage) Photolysis of H2OHow does the indirect photolysis system work?
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Engineering Metabolic Pathways in Heterocysts
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Engineering Metabolic Pathways in Heterocysts
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Hydrogen Measurement: Hydrogen Analyz
GC
Gas sample
Reduction Detector: HgO + H2 Hg (g)
Measured by Spectrophotometer
Batch Cultures
Hydrogen Analyzer
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0
10
20
30
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60
70
80
0 25 50 75 100
Time (hr)
H2
( µm
ols)
WT
∆hyaB
∆hydA
∆hydA / ∆ hyaB
0.01
0.10
1.00
0 25 50 75 100
Time (hr)
OD
(660
nm
)
WT
∆hydA / ∆ hyaB
∆hydA
∆hyaB
Hydrogen evolution in S. oneidensis MR-1 wild type and mutants
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Loss of hydrogen evolution fromAnabaena 7120 after culturing on
nitrogen repleted medium for extendedperiod of time
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http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG
Oxygenic Photosynthesis: Light Reaction
H2
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Direct Cyanobacterial Hydrogen Production
2 H2O
O2+ 4 H +PS II PS I
hνhν
ReducedFerredoxin
NADP +
NADPH
Synechocystis Catabolism
Glucose 2 Pyruvate
2 Acetyl CoA+ 2 CO 2
2 ReducedFerredoxins
Hydrogenase2 H+
H2Clostridium pasteurianum Catabolism
New Pathway
Sunlight
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BiomassBioenergy Crop Biomass Residues
H2
CO 2
CO 2
Biomass
Gasification
H2
CO 2
CO 2
Biomass
GasificationFood
Wood
H2OH2OBiomassBiomass
Direct photobiological H2 production
H2O
H2 O2
Biomass
Biomass
Source: NAS/NRC 2004
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Chlamydomonas reinhardtii (Algal ) H2 Production System:
Sulfur Stress PSII degradation Anaerobiosis H2ase induction
Melis & Happe. 2001. Plant Physiol: 127:740.
http://www.tigr.org/tdb/images/chrgi.gif
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Our Goal:To develop a new biological system to extract hydrogen from water (Biohydrogen). Features of this system include:
-Driven by sunlight-Renewable (Sunlight + Water)-Can be performed under ambient air condition (Does not requireanaerobic condition-Higher photo-conversion efficiency (Light energy Hydrogen)-Minimal impact on the environment (Low nutrient input, Use of GMO)-Inducible
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Limitations of currently available systems for Biohydrogen production (For Example, the Algal/Chlamydomonas system):
-Nearly all require anaerobic condition during hydrogen production-Low photo-conversion efficiency-High cost -Discontinuous production (Initial biomass accumulation phasefollowed by the anaerobic hydrogen production phase)
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The low efficiency is due to the inherent conflicts involving the 2 biochemical processes (Oxygenic Photosynthesis and Hydrogen Evolutio
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The solution:
Separate Photosynthesis and Hydrogen Evolutionspatially:
Indirect (2-Stage) Photolysis of H2O
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H2 Chromatograph:
H2 Peak
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Indirect (2-Stage) Photolysis of H2OGoal: Photosynthesis and hydrogen production occurs in spatially separate compartments (cell types).
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Simplified version of biochemicalpathways in (A) normal vegetative
cells and heterocysts and in (B)modified heterocysts for hydrogen
production.