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.