Post on 14-Nov-2020
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
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
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
Oxygen sensitivity of Hydrogenases
2H+
[e-] (reduced Ferredoxin)
H2O2
http://www.wright.edu/biology/faculty/carmichael/labhome/green.jpg
The microorganisms of choice: Nitrogen-fixing Filamentous Cyanobacteria
MH
PH
Fluorescence Images of Vegetativecells and Heterocysts
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG
Light Reaction in Heterocysts
Function of heterocysts in filamentous cyanobacteria
O2
N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi
N2-ase reaction
Engineering heterocysts forcyanobacterial hydrogen production
Engineered Anabaena
Anabaena wild type
Plasmid for shydA expression
H2 Chromatogram:
H2 Peak
Relative transc ript levels o f nifD and shydA
Relative transc ript levels o f nifD and shydA
sHy dA protein leve l
Integration of shydA into the nifHDKoperon in Anabaena 7120
Hydrogen production from Anabaena7120 ex-conjugant
Assembly and Maturation of Fe-only Hydrogenases
Alfred M. Spormann, Galit Meshulam-Simon, James R. Swartz
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
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+)
Acknowledgements• Jim Swartz• Chia-Wei Wang
• Peter Wolk (MSU)• Terry Thiel (UM, St.
Louis)
Funding:
Stanford SOE seed fund
Fe-only Bi-directional Hydrogenases
2H+
[e-] (reduced Ferredoxin)
H2
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
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
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
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
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
Solar Energy
Microorganisms, Plants
O2CO 2H2O Biomass
Biological Ecosystems
Fossil Fuels
Solar Energy
Sustainable clean energyFuel cell engines
H2O O2 H2
Engineered Microorganisms
H2O
Photons (h ν )
PS II+PS I 2[H] + 1/2 O 2 O 2
PhysiologicalPath
Cell Mass
CO2
H2
EngineeredPath
Filamentous Cyanobacteria
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
www.chem.cmu.edu/.../ achim/research/magneto.html
Nitrogenase reactionO2
N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi
Programmed Heterocyst Differentiation
Indirect (2-Stage) Photolysis of H2OHow does the indirect photolysis system work?
Engineering Metabolic Pathways in Heterocysts
Engineering Metabolic Pathways in Heterocysts
Hydrogen Measurement: Hydrogen Analyz
GC
Gas sample
Reduction Detector: HgO + H2 Hg (g)
Measured by Spectrophotometer
Batch Cultures
Hydrogen Analyzer
0
10
20
30
40
50
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
Loss of hydrogen evolution fromAnabaena 7120 after culturing on
nitrogen repleted medium for extendedperiod of time
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG
Oxygenic Photosynthesis: Light Reaction
H2
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
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
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
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
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)
The low efficiency is due to the inherent conflicts involving the 2 biochemical processes (Oxygenic Photosynthesis and Hydrogen Evolutio
X
The solution:
Separate Photosynthesis and Hydrogen Evolutionspatially:
Indirect (2-Stage) Photolysis of H2O
H2 Chromatograph:
H2 Peak
Indirect (2-Stage) Photolysis of H2OGoal: Photosynthesis and hydrogen production occurs in spatially separate compartments (cell types).
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG
Simplified version of biochemicalpathways in (A) normal vegetative
cells and heterocysts and in (B)modified heterocysts for hydrogen
production.