PRINCETON PENNSTATE HAWAII Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs AFOSR MURI Progress Update: January 2007 -BioSolarH 2 Team Charles Dismukes PU photosyn. metabolism/chemistry Donald A. Bryant PSU genetics/mol biology cyanos Matthew Posewitz CSM genetics/mol biology algae Eric Hegg MiSU enzymology/inorganic Robert Bidigare UH microbial physiology/ecology >2006 John Peters MoSU crystallography & hydrogenases Robert Austin PU microchemostats & cellular evolution http://www.princeton.edu/~biosolar/
Native & GMOs that produce more energy -BioSolarH 2 The Approach: Algal & Cyanobacteria cell factories that produce chemical energy w/o sacrificing the microbe Sunlight CO 2 H 2 0 Nutrients Cell Factories Chemical energy H 2, alcohols, acids, hydrocarbons
The Goal 2 H 2 O O 2 + 2 H 2 (H + /e + )
Rapid H 2 & Fluorescence Screening CSM/NREL H 2 gas sensor Microarrayed chemostats for directed evolution Fast repetition rate fluorimeter Instrumentation Development Metabolomics & Proteomics Dissolved H 2 & O 2 LED + Clark cells Two LC-MS Engines Active February 2007
Screening Sampling of Culture Collections in Progress H 2 production Photosynthetic Quantum Efficiency Marine Microbial Ecology and Diversity Center, University of Hawaii Patterson Collection. Largest collection of cyanobacteria in the world Approx. 1800 viable strains Difficult to assemble a library of this scope due to sampling rights. NREL Collection. 180 strains collected from the US southwest for NREL biodiesel program. Mitsui collection. 165 strains marine cyanobacteria. Actively expanding collection Bioprospecting Great Salt Lake & Yellowstone NP
Why Screen? Jonathan Meuser & Gennady Ananyev Screening has revealed major differences in photo-h 2 production and rates of anaerobic induction between ecologically distinct strains of monophyletic genera of algae Microalga Chlamydomonas The time profiles reveal the kinetics of induction of photo-h 2 production capacity that occurs following initiation of anaerobisis at time zero of cultures grown on light photosynthetically. Six strains of Chlamydomonas
Micronutrients, growth & bioreactor optimization has produced large improvements in H 2 production 1. Sufficiency of Nickel during growth 17X H 2 increase 2. Duration of prior photoautotrophic growth. oldies but goodies 3. Anaerobicity & darkness during fermentation. strong respiring strains 4. Selection of salt-tolerant strain. synthesis of fermentable sugars 5. Mechanical agitation used for photoautotrophic growth. minimize shearing 6. Higher fermentation temperature. 2x (23 30 C) 7. Lower light intensity 3x after attaining steady-state. Fermentation adaptation
Ni 2+ Supplementation on Growth & H 2 Production by A. Maxima Damian Carrieri & Gennady Ananyev A. [Ni 2+ ] causes chlorosis during initial stage of photoautotrophic growth (130µE/m 2 sec). C. Ni 2+ stimulates cell s capacity to evolve hydrogen by 20 fold! B. After chlorosis induced lag, cells 10 recover & grow photoautotrophically to normal cell density. NO Effect. Chl Absorption Time (Hours) 1 0 200 400 600 800 1000 0.0uM Ni 0.1 0.25uM Ni 1.0uM Ni um Ni 4.0 MV (reduced) + 2H + (aq) MV (oxidized) + H 2 0.01
2006 rates & yields of H 2 by cyanobacteria & algal strains Future target Cell free H 2 ase 28 ml H 2 L -1 h -1 H 2 build-up, 30 C H 2 vented, 23 C In light In Dark 2006 NREL immobilized algae * 0.35% photo-h 2 by C. reinhardtii under Sulfur depletion Ghirardi, Seibert, et al. (2004, 2006) 2004 NREL batch algae* 0.15% 2006 Arthrospira Maxima H 2 yield in the dark 250 ml H 2 L -1 in 4 days batch culture, 1 growth cycle. % Efficiency Solar-to-H 2 without Nickel
Osmotic Stress by Dilution of Growth Medium Boosts H 2 Yield by 18 X! Arthrospira maxima acclimated to NaCl Dilution of salt 250 mm Carbonate medium + 250 mm NaCl +500 mm NaCl +750 mm NaCl
Arthrospira exhibits large increases in fermentative H 2 production by application of stresses or by carbon supplementation : 1. Osmotic stress of salt-tolerant strains. 18X increase 2. Nitrate removal during fermentation (if sole N source). > 5-20X 3. Add fermentable carbon substrates. > 10X with glucose These results with the Princeton microbioreactor indicate promising Applications for larger scale fermentation trials in progress
Genetic Opportunities Ahead Develop a genetic system for Arthrospira sp. Determine the optimal conditions for H 2 ase expression in cyanobacteria Over-express H 2 ase(s) in cyanobacteria Introduce foreign H 2 ase(s) into cyanobacteria [NiFe]-hydrogenase engineering of NAD vs NADP selectivity. [NiFe]-hydrogenase gene shuffling Engineer cyanobacterial strains with reduced amounts of light-harvesting antenna Use comparative genomics, molecular genetics, and strain engineering to optimize H 2 production
Obtain genome sequences from robust, hydrogen-producing oxyphototrophs Synechococcus sp. PCC 7002 Arthrospira maxima Develop genetics in this excellent H 2 -producer; genome will be sequenced in 2007 by JGI-DOE Genome Apply -omics Transcriptomics Proteomics Metabolomics Metabolic Engineering hoxe hoxf hoxu hoxy hoxh H 2 hype hyp3 hoxwhypahypb hypf hypc hypd In Synechococcus sp. PCC 7002, the genes encoding the Ni-Fe hydrogenase uniquely form an operon of 13 genes
Improving H 2 production with proven genetics tools Full genome available for Synechococcus sp. PCC 7002 Chromosome paq1 paq4 paq3 paq5 paq7 Identify, and then delete or inactivate, genes for pathways that compete for electrons with H 2 ase; enhance glycogen storage paq6 Synechococcus sp. PCC 7002 strains for enhanced H 2 production LIGHT grow 40.2 Over-express genes for modified hydrogenases ferment H 2 40 33.4 30.9 paq1 paq3 20 16.1 16.9 11.1 0 6.7 3.8 7.9 6.9 6.9 Chromosome paq1 paq3 paq4 paq5 paq6 paq7 3.7 6.0 3.6 Insert hydrogenase structural genes in paq1 Insert hydrogenase accessory genes in paq3 OD550::0.7 OD550::3.0 Actual copy numbers for chromosome and plasmids in Synechococcus sp. PCC 7002 cells in midexponential and early stationary phase
3 Fermentative Gene Knockout Targets based on Genome Sequences glycogen G6P Kelsey McNeely & Bryant Lab H + H + H + NAD(P)H Fd: NADP H + 2 ase oxidoreductase NAD(P) + H 2 acetyl phosphate ADP acetate kinase ATP CH 3 COO Fd (ox) Fd (red) phosphotransacetylase Acetate acetyl-coa synthetase 2 NAD + + 2 ADP, P i glycolysis pyruvate pyruvate:fd oxidoreductase 2 NADH + 2 ATP D-lactate dehydrogenase NAD(P)H acetyl-coa+ CO 2 NAD(P)H aldehyde dehydrogenase NAD(P) + acetaldehyde NAD(P)H alcohol dehydrogenase NAD(P) + CH 3 CH 2 OH Ethanol NAD(P) + OH O Synechocystis 6803 only O D-lactate Synechococcus 6803 & 7002
In vitro Hydrogenase activation: a robust platform to investigate hydrogenase maturation In vitro activation of heterologously expressed hydrogenase structural protein HydA (HydA ΔEFG ) by the addition of co-expressed accessory proteins HydE, HydF and HydG. A robust system amenable to in depth study and characterization of hydrogenase maturation. The hydrogenase H cluster In vitro activation is the first step in the characterization of the biochemical reactions involved in H Cluster biosynthesis and hydrogenase maturation. These studies are critical to the effective genetic engineering of organisms expressing [FeFe] hydrogenase
Analysis of C. reinhardtii transcriptome under H 2 producing conditions Levels of over 500 transcripts change significantly. Several hundred are of unknown function. Novel targets potentially influencing hydrogenase activity have been identified. Analysis of the transcriptome during H 2 production is essential to understand hydrogenase activity in the context of whole-cell metabolism. Pathways of electron transfer are being analyzed and targets to enhance H 2 production identified.
Application of gene-shuffling for the rapid generation of novel [FeFe]- hydrogenase libraries Gene shuffling protocol was identified, optimized and used to rapidly generate libraries of unique [FeFe]- hydrogenases Generates a high percentage of active enzymes in E. coli User friendly and requires a single set of maturases CaHydA1 CsHydA1 LEN-24 LEN-23 LEN-22 LEN-21 LEN-20 LEN-19 LEN-18 LEN-17 LEN-16 LEN-15 LEN-14 LEN-13 LEN-12 LEN-11 LEN-10 LEN-9 LEN-8 LEN-7 LEN-6 LEN-5 LEN-4 LEN-3 LEN-2 LEN-1 0 60 120 180 240 300 360 420 480 540 600 660 Protein length (AA)