Biotechnology for renewable energy and environmental protection Kornél KOVÁCS Dept. Biotechnology, Univ. Szeged Institute of Biophysics, BRC

BIOHYDROGEN

Need for alternative energy Fossil energy shortage Oil and natural gas: <30 years Coal: greenhouse effect Fossil energy Coal Oil and natural gas yr

Nukleáris energia: maghasadás / magfúzió Nuclear energy: nuclear fission / fusion Coal Wood Natural gas Oil Fission Solar yr

HYDROGEN METABOLISM Archeae, Eubacteria, Cyanobacteri, Algaea Enzymes Nitrogenase (N2 + 4H2  2 NH4+) Hydrogenase (2H+ + 2e-  H2) Fe-only (e.g. Clostridium) [NiFe] No metal (methanogen)

Solar energy numbers Energy utilization today 1 Known fossil reserves 100 Coal ~ 80 Oil, natural gas ~ 20 Solar energy on Earth 10000 1 hour of Solar  1 year of use today

Several sources, one carrier: H2

BIOLOGICAL H2 PRODUCTION Hydrogenase Photosynthesis e- + H2O O2 2e- 2 H+ H2 2H+ Biomass e- 2 H+ H2

HYDROGEN METABOLISM Archeae, Eubacteria, Cyanobacteria, Algaea Enzymes Nitrogenase (N2 + 4H2  2 NH4+) Hydrogenase (2H+ + 2e-  H2) Fe-only (e.g. Clostridium) [NiFe] No metal (methanogen)

MICROORGANISMS Thiocapsa roseopersicina Thermococcus litoralis, T. sibiricus Methylococcus capsulatus (Bath) Methylocaldum szegediense

HYDROGENASES Redox metalloenzymes Conserved sequence and structure [NiFe] + n [Fe4Sx] Conserved sequence and structure Very sensitive heat, oxygen, redox potential Biotechnology needs stable catalyst !!! H2 evolution  biohydrogen H2 uptake  fuel cell

Hydrogenase activity assay

Biohydrogen utilization evolution reduction ENERGY hydrogenase H2 uptake oxidation e- REDUCINGAGENT Biogas, denitrification, etc.

[NiFe] hydrogenases Heterodimer [NiFe] + CO/2CN 3 x [Fe4S4] 64kDa + 34kDa [NiFe] + CO/2CN on large subunit 3 x [Fe4S4] on small subunit 14-15 Angstrom apart

Thiocapsa roseopersicina BBS - phototrophic purple sulfur bacterium - max. growth temperature 30°C - anaerobic growth - isolated from cold sea water - [NiFe] hydrogenases H2  2H+ + 2e-

T. roseopersicina stable Hase  pure enzyme  whole cell Heat Oxygen Proteases Specific activity Temperature

LOCATION: Hyd / Hup / Hox Xred: GASH ? 2H+ PS ATP ? H2 HydL 2H+ 2e- Xox Xred ADP+Pi ATP HydS Isp1 Isp2 2e- + 2H+ N2ase 2H+ ATP ADP+Pi NH4+ 2e- N2 HupL HupS HupC 2H+ HoxH HoxY HoxU HoxF NAD+ NADH+H+ GASH:glutathion amide

Hydrogenase inventory Reaction catalysed: H2  2H+ + 2e- Stable Hase in the membrane: heat, protease hydS hydL isp2 isp1 hupS hupL hupC hupD hupH hup I hupR Unstable Hase in the membrane: why unstable? Hase in the cytoplasm: hydrogen evolution? hoxF hoxH hoxY huxU Hydrogen sensing Hase: oxygen, CO hupT hupU hupV Thiocapsa roseopersicina

Interposon mutagenesis of the hydrogenase structural genes sacB hydS isp1 isp2 hydL Sm strain:GB11 hupS hupL hupC hupD hupH hupI ORF-1 hupR sacB Gm strain: GB1121

GB1121: The first good H2 producing strain by hydrogenase enzyme 140 120 100 80 In vivo H2 production (%) 60 40 20 wild type double mutant HypF-mutant strains Non nitrogen-fixing conditions -there is at least one additional hydrogenase in Thiocapsa roseopersicina !

? In the GB1121 strain the H2 is produced by hydrogenase citoplasm PS ATP 2H+ H2 2H+ HydL HupL HydS HupS Isp1 2e- HupC Isp2 ADP+Pi ATP ADP+Pi NH4+ ATP 2e- 2e- + 2H+ ? N2 Xred Xox H2 N2ase Xred: GASH ? H2 citoplasm GASH: glutathion amide 2H+ soluble hydrogenase

3D MODELLING Only in HydSL Only in HupSL-ben Shared

Looking for stabilizing elements Random mutagenesis PCR errors (labor evolution) Chimeric enzymes DNS shuffling Site specific mutagenesis

Accessory genes R. capsulatus hup: W X T U V S L C D FG H J K R hyp: F A B D E

Applied gene transfer systems E. coli : donor T. roseopersicina: recipient mob Tn5 RP4 tra Site directed mutagenesis Normal conjugation Transposon Complementation

Screening H2 H2 + 2MV2+ 2MV+ + 2H+ Hydrogenase Heat treatment (optional) methyl-viologen 75 oC, 1-3h, air H2 H2 + 2MV2+ 2MV+ + 2H+ Hydrogenase

Mutagenesis studies Hase maturation = complex process Investigation: - mutation = loss of function - correction via complementation Wild Mutant hypF hupK hypC1 hypD hypE hypC2 hydD

polypeptide (HupL/HydL) Maturation / Assembly Large subunit polypeptide (HupL/HydL) Small subunit polypeptide (HupS/HydS) folding HypC Fex-Sx incorporation Fex-Sx cluster synthesis HypE, Fe? HypF (CO, CN) HypB (Ni) HypD HypD + HypC C-terminal peptidase (HupD/HydD) Membrane transport Tat-mechanisms active enzyme

Role of the HypC protein in the maturation process Small subunit C Cys Large subunit HypC

Role of the HypF auxiliary protein Cys HypC N C Cys Large subunit N Small subunit CN- CO HypF CN- (+HypE)

Role of the HypB and HypD proteins GTP GDP + Pi Ni HypB Cys HypC N C Cys Large subunit N Small subunit CO CN Fe HypD

In vivo hydorgen production (%) In vivo H2 production 700 600 500 400 In vivo hydorgen production (%) 300 200 100 wild type double mutant hypF- Nitrogen-fixing conditions

polypeptide (HupL/HydL) Maturation / Assembly Large subunit polypeptide (HupL/HydL) Small subunit polypeptide (HupS/HydS) folding HypC Fex-Sx incorporation Fex-Sx cluster synthesis HypE, Fe? HypF (CO, CN) HypB (Ni) HypD HypD + HypC C-terminal peptidase (HupD/HydD) Membrane transport Tat-mechanisms active enzyme

HypF- = Hyd- / Hup- / Hox- N2áz 2e- + 2H+ 2H+ ATP ADP+Pi NH4+ 2e- N2 HupL HupS HupC H2 HydL Xred HydS Isp1 Isp2 Xred: GASH ? PS ? GASH:glutation amide Nitrogenase Hydrogen is produced by the nitrogenase complex

Effect of the hupK deletion on the Hyd uptake activity 100 100 80 activity % 60 40 20 4,7 HupK- mutant wild type -measured on membrane fraction at 55 oC

Accessory genes R. capsulatus T. roseopersicina hup: W X T U V S L C D FG H J K R hyp: F A B D E T. roseopersicina (instabil) : struktural genes (hupS, hydS, hupL, hydL) : accessory genes in R.capsulatus : accessory genes in T. roseopersicina : accessory genes in both : pleiotrop genes (hyp) hyd: hup: (stabil) isp1 isp2 S L S L C D H I R hyp F C D E hup K hyd D

BioHyd EU project 2. óra anyaga itt! klikk!!