hydA - Stanford University
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Metabolic Engineering of Hydrogen Production in Filamentous Cyanobacteria Alfred M. Spormann, Wing-On (Jacky) Ng Departments of Civil & Environmental Engineering, of Biological Sciences, and of Geological & Environmental Sciences Stanford University September 19, 2006 Photons (hν ) H2O PS II+PS I Physiological Path CO 2 Cell Mass O2 2[H] + 1/2 O2 Engineered Path Hydrogen formation and oxygen evolution are naturally incompatible H2 Photons (hν ) H2O PS II+PS I Physiological Path O2 2[H] + 1/2 O2 Engineered Path Hydrogen formation and oxygen evolution are naturally incompatible H2 Oxygen sensitivity of Hydrogenases 2H+ O2 H2 [e-] (reduced Ferredoxin) The microorganisms of choice: Nitrogen-fixing Filamentous Cyanobacteria http://www.wright.edu/biology/faculty/carmichael/labhome/green.jpg Fluorescence Images of Vegetative cells and Heterocysts MH PH Light Reaction in Heterocysts http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG Function of heterocysts in filamentous cyanobacteria N2-ase reaction O2 N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi Engineering heterocysts for cyanobacterial hydrogen production Anabaena wild type Engineered Anabaena Plasmid for shydA expression H2 Chromatogram: H2 Peak Relative transc ript l evels o f nifD and shyd A Relative transc ript l evels o f nifD and shyd A sHy dA prot ei n leve l Integration of shydA into the nifHDK operon in Anabaena 7120 Hydrogen production from Anabaena 7120 ex-conjugant Assembly and Maturation of Fe-only Hydrogenases Alfred M. Spormann, Galit Meshulam-Simon, James R. Swartz The active site of Fe-only hydrogenase The 6.8 kb SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 SO3925 Hypothetical hydE SO3926 hydF Helper proteins involved in HydA maturation - Role of HydGEX SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 l hydX SO3925 hydE SO3926 hydF µmol H2/ OD 200 150 100 50 0 ∆hyaB (hydA+) ∆hyaB/∆hydG ∆hyaB/∆hydE ∆hyaB/∆hydX 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+ H2 [e-] (reduced Ferredoxin) Helper proteins involved in HydA maturation - Role of HydG SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 l hydX SO3925 hydE SO3926 hydF 400 Active HyaB 350 µmol H2 / OD 300 250 Active HydA 200 150 100 50 0 wt WT ∆hydA ∆hydA ∆hydA∆hydG ∆hydA/∆hydG ∆hyaB ∆hyaB∆hyaB∆hydG ∆hyaB/∆hydG Helper proteins involved in HydA maturation - Role of HydE SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 l hydX SO3925 hydE SO3926 hydF 400 350 µmol H2 / OD 300 250 200 150 100 50 0 wtWT ∆hydA ∆hydA ∆hydA∆hydE ∆hydA/∆hydE ∆hyaB ∆hyaB∆hyaB∆hydE ∆hyaB/∆hydE Helper proteins involved in HydA maturation - Role of HydX SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 hydX SO3925 hydE SO3926 hydF 400 350 µmol H2 / OD 300 250 200 150 100 50 0 wtWT ∆hydA ∆hydA ∆hydA∆hydX ∆hydA/∆hydX ∆hyaB ∆hyaB ∆hyaB∆hydX ∆hyaB/∆hydX Helper proteins involved in HydA maturation - Role of HydE SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 l hydX SO3925 hydE SO3926 hydF 400 350 µmol H2 / OD 300 250 200 150 100 50 0 wtWT ∆hydA ∆hydA ∆hydA∆hydE ∆hydA/∆hydE ∆hyaB ∆hyaB∆hyaB∆hydE ∆hyaB/∆hydE Helper proteins involved in HydA maturation - Role of HydX SO3920 SO3921 hydA hydB Large subunit Small subunit SO3922 fdh SO3923 hydG SO3924 hydX SO3925 hydE SO3926 hydF 400 350 µmol H2 / OD 300 250 200 150 100 50 0 wtWT ∆hydA ?hydA ∆hydA/∆hydX ?hyaB ∆hyaB ?hyaB?hydX ∆hyaB/∆hydX ?hydA?hydX Solar Energy Microorganisms, Plants H 2O CO 2 Biological Ecosystems Fossil Fuels O2 Biomass Solar Energy Engineered Microorganisms O2 H 2O Sustainable clean energy Fuel cell engines H2 Photons (h ν ) H2O PS II+PS I Physiological Path CO 2 Cell Mass O2 2[H] + 1/2 O 2 Engineered Path H2 Filamentous Cyanobacteria Unique biological adaptation of filamentous cyanobacter Anabaena sp. Heterocysts (Anaerobic, N2-Fixation) Vegetative cells (Oxygenic Photosynthesis) Source: http://web.uvic.ca/~h2o/Phyto2/Anabaena_affinis.jpg Nitrogenase reaction O2 N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi www.chem.cmu.edu/.../ achim/research/magneto.html Programmed Heterocyst Differentiation Indirect (2-Stage) Photolysis of H2O How does the indirect photolysis system work? Engineering Metabolic Pathways in Heterocysts Engineering Metabolic Pathways in Heterocysts Hydrogen Measurement: Hydrogen Analyz Gas sample Batch Cultures Reduction Detector: HgO + H2 Æ Hg Hydrogen(g)Analyzer GC Measured by Spectrophotometer Hydrogen evolution in S. oneidensis MR-1 wild type and mutants OD (660 nm) 1.00 ∆ hydA WT 0.10 ∆ hyaB ∆ hydA / ∆ hyaB 0.01 0 25 50 75 100 Time (hr) 80 WT 70 ∆ hydA H2 ( µmols) 60 ∆ hyaB 50 40 30 20 10 ∆ hydA / ∆ hyaB 0 0 25 50 Time (hr) 75 100 Loss of hydrogen evolution from Anabaena 7120 after culturing on nitrogen repleted medium for extended period of time Oxygenic Photosynthesis: Light Reaction H2 http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG15_08.JPG Direct Cyanobacterial Hydrogen Production Sunlight hν hν 2 H2O PS II PS I O2+ 4 H + Reduced Ferredoxin NADP + NADPH SynechocystisCatabolism New Pathway Glucose 2 Pyruvate 2 Acetyl CoA + 2 CO 2 2 Reduced Ferredoxins Hydrogenase ClostridiumpasteurianumCatabolism 2 H+ H2 Biomass BioenergyCrop Biomass Residues Biomass CO 2 Direct photobiological H2 production Biomass H2O CO 2 Biomass H2O Biomass H2O Biomass Biomass Gasification Food Wood H2 CO 2 Source: NAS/NRC 2004 Gasification H2 CO 2 H2 O2 Chlamydomonas reinhardtii (Algal ) H2 Production System: http://www.tigr.org/tdb/images/chrgi.gif Sulfur StressÆPSII degradationÆAnaerobiosisÆH2ase induction Melis & Happe. 2001. Plant Physiol: 127:740. 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 require anaerobic condition -Higher photo-conversion efficiency (Light energy Æ 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 phase followed 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 Evolution spatially: Indirect (2-Stage) Photolysis of H2O H2 Chromatograph: H2 Peak Indirect (2-Stage) Photolysis of H2O Goal: 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 biochemical pathways in (A) normal vegetative cells and heterocysts and in (B) modified heterocysts for hydrogen production.
