Hydrogen from Water in a Novel Recombinant Oxygen-Tolerant Cyanobacterial System Philip D. Weyman, Isaac T. Yonemoto, and Hamilton O. Smith, J. Craig Venter Institute Key Collaborator: Pin-Ching Maness, National Renewable Energy Laboratory Project ID PD039 This presentation does not contain any proprietary, confidential information, or otherwise restricted information Overview Timeline Barriers . Project start date: 5-01-2005 . Barriers addressed . Project end date: 1-30-2014 – Biological Hydrogen . Percent complete: 91% Production Barrier AP: Budget Oxygen Accumulation . Total project funding – DOE share: $2.019M for JCVI Partners – DOE share: $1.26M for NREL . – JCVI cost-share: $820K National Renewable Energy . Funding received for FY12 Laboratory – $200K for JCVI . Planned funding for FY13 – $150K for JCVI Slide 2 Relevance: Hydrogenase + Develop an O2- H H2 tolerant cyanobacterial Energy for system for normal metabolism Ferredoxin continuous light- driven H2 Membrane production from Photosystem I water + H2O H + O2 Photosystem II Barrier AP: O2 Accumulation 2011 Status 2015 Target 2020 Target Duration of continuous photoproduction in full sunlight 2 min 30 min 4 h Slide 3 Approach: Milestones and Go/No Go Task 1. Engineering known hydrogenases Month/ Milestone % Comp Year Sept-11 Purify hydrogenase and verify functionality in O2 100% Dec-10 Determine electron mediator requirement 100% Sept-11 Verify hydrogenase activity in cyanobacteria in vivo and ability to make H2 from water 100% Task 2. Discovery and engineering of new hydrogenases Month/ Milestone % Comp Year 100% Sept-10 Identify novel hydrogenases from environment and transfer to cyanobacteria 100% Aug-11 Construct a cyanobacterial hybrid to express an active environmental hydrogenase Increase activity of HynSL hydrogenase in cyanobacteria to give 100-fold increase in 30% Apr-13 specific activity. Improve hydrogenase-ferredoxin (Fd) electron transfer to enable 25-fold better Fd 20% Nov-13 docking to the hydrogenase. Jan-14 Measure light-dependent H2 production in modified strains. 5% Go/No Go Decision: Due Jan-13, Achieved Nov-12 Demonstrate 5x increase hydrogenase activity from environmental H2ase in cyanobacteria as measured by in vitro H2 evolution assay. Slide 4 Task 1: Technical Approach Transferring a known O2-tolerant NiFe-hydrogenase from T. roseopersicina into cyanobacterium Synechococcus sp. PCC 7942 O2-tolerant Hydrogenase + H H2 O - O - 2 2 Gene tolerant tolerant NiFe- NiFe- transfer H2ase H2ase Ferredoxin Membrane Recombinant T. roseopersicina Photosystem I + Cyanobacterium H2O H + O2 Photosystem II Slide 5 Task 1: Technical Approach Review from previous AMR: 0.1 • Completed milestone 0.08 “Construct cyanobacterial 0.06 hybrid to express active 0.04 Thiocapsa hydrogenase” evolution assay evolution /mg protein/h 2 2 0.02 (8/11). H 0 • Effort in FY2011-13 focused on increasing expression of nmole In In H vitro heterologous hydrogenase using environmentally-derived hydrogenase as model. JCVI approach is complementary to that of NREL in harnessing Nature’s O2-tolerant hydrogenases and their transfer into two model cyanobacteria. Slide 6 Task 2: Technical Approach Task 2. Identifying novel O2-tolerant hydrogenases through metagenomic analysis of marine microbes in the global ocean and transferring the hydrogenases into cyanobacteria Sorcerer II Expedition: Global Ocean Sampling Project (Funded as cost share at no expense to DOE) . This approach is complementary to two approaches in the Task 1 about harnessing nature’s O2-tolerant hydrogenases and their transfer into cyanobacteria. Slide 7 Task 2: Technical Approach • Previously, we reported transfer of 5.0 hydrogenase from environmental DNA to 4.0 Synechococcus PCC 7942 (Top figure, 3.0 Milestone reached 4/10) 2.0 protein/h • Environmental hydrogenase is more 1.0 nmole H2/mg thermostable and O2-tolerant than 0.0 Thiocapsa HynSL. WT +IPTG Empty pRC41 Vector +IPTG +IPTG • Cyanobacterial ferredoxin (PetF*) can act 0.2 as an electron mediator to the /mg environmental hydrogenase in E. coli 2 crude extract (Bottom figure, Milestone H 0.1 “Determine Electron Mediator protein/h Requirement” reached 12/10) nmole 0.0 * PetF cloned and purified by NREL Empty Vector pRC41 (Dithionite pRC41 (Dithionite+PetF) only) (Dithionite+PetF) and provided to JCVI through our collaboration Slide 8 2 Current Approaches to Improve System 1. Improve expression and activity of hydrogenase in cyanobacteria (Task 2.4) 2. Improve hydrogenase-ferredoxin interaction (Task 2.5) Hydrogenase + H H2 Energy for normal metabolism Ferredoxin Membrane Photosystem I + H2O H + O2 Photosystem II Slide 9 Task 2: Technical Approach Review from last year’s AMR: • More Trc promoters increases hydrogenase expression • Ratios of maturation proteins may be important for maturation efficiency • Promoters at A+D led to more “mature” HynL but no increase in activity A B C D Trc Promoters: A A+D A+B+C+D Slide 10 Task 2: Technical Accomplishments Testing promoter placement, strength, and frequency • Plan of promoter placement = 27 different variants • Relative activities of promoters we are using 1000 100 activity Relative promoter promoter Relative 10 no promoter RBC TRC PSBA control On track to complete Milestone “Increase activity of HynSL hydrogenase in cyanobacteria to give 100-fold increase in specific activity.” Slide 11 Task 2: Technical Accomplishments Engineer T7 polymerase strategy for hydrogenase expression. Potential Advantages of T7 polymerase: • Tighter control of transcription • Lower frequency of early termination, may function better with long transcripts. 9 8 A 7 6 B 5 4 C 3 2 D Fold induction by nitrate by induction Fold 1 (normalized to cell to cell (normalized density) 0 0 5 10 15 20 Hours after wash Progress toward Milestone “Increase activity of HynSL hydrogenase in cyanobacteria to give 100-fold increase in specific activity.” (4/13). Slide 12 Task 2: Technical Approach • FeS clusters form a molecular wire to/from the active site Slide 13 Task 2: Technical Approach Point mutants alter electrochemistry of the hydrogenase small subunit “molecular wire” Large subunit Catalytic center FeS cluster variants 1 2 3 4 Electron Iron-sulfur transfer clusters interface Small subunit Tests the potential to further modify the environmental hydrogenase to favor H2 production in vivo using PetF as an electron mediator. Slide 14 Task 2: Technical Approach Review from last year’s AMR: Double-substituted HynS yields increased evolution activity relative to uptake Total protein (Sypro® Ruby stain) αHynL EV 1 2 3 4 Slide 15 Task 2: Technical Accomplishments Increased H2 evolution in Crude Purified construct 4 is also observed in purified protein HynS- HynS- Strep Strep Specific activity (nmole H2 1 4 1 4 1 4 produced/mg Fold protein/h) purification αHynL Construct 1 (crude) 369 Construct 1 (purified) 41,450 112 Construct 4 (crude) 765 αStrep=(HynS) Construct 4 (purified) 134,214 176 Total protein Completed Milestone 2.5.1 “Develop an (Sypro Ruby affinity-tagged purification system for stain) HynSL” (08/12). Slide 16 2 Current Approaches to Improve System 1. Improve expression and activity of hydrogenase in cyanobacteria (Task 2.4) 2. Improve hydrogenase-ferredoxin interaction (Task 2.5) Hydrogenase + H H2 Energy for normal metabolism Ferredoxin Membrane Photosystem I + H2O H + O2 Photosystem II Slide 17 Engineering cycle for improving H2ase activity H2 Test Design E. coli Frontline work Synechococcus Build Fast elongatus Best candidates Slow Slide 18 Task 2: Technical Accomplishments Wild type Mutant HynS mutants with altered ferredoxin binding sites retain activity Key: Red = negative charge Blue = positive charge On track to complete Milestone “Improve hydrogenase-ferredoxin (Fd) electron transfer to enable 25-fold better Fd docking to the hydrogenase.” (11/13). Slide 19 Task 2: Technical Accomplishments Ferredoxin-fusion H2ases are active when expressed in E. coli 14 Immunoblot with anti- 12 Strep antibody 10 Fusion 55 8 (44kD) 37 6 Immat lysate/min) HynS 4 25 (32kD) Production (nmol/mg (nmol/mg Production 2 2 H 20 ? 0 CM012 4 pIY086 5 pIY103 6 15 Double- DS HynS DS HynS + Substituted + Strep ferredoxin 4 5 6 HynS tag + Strep tag On track to complete Milestone 2.5.3 “Construct an Fd-HynS fusion protein” (11/13). Slide 20 Task 2: Technical Accomplishments Activity improvements can be combined 45 40 Key: 35 A = Envi H2ase 30 B = A + extra 25 promoters 20 /mg protein/h 2 H 15 C = B + Double 10 nmol substitution 5 HynS 0 WildWT HoxmutNo RC41A RC41-4B CM12C type H2ase Exceded Go-No Go Criteria “Demonstrate 5x increase hydrogenase activity from environmental H2ase in cyanobacteria as measured by in vitro H2 evolution assay.” (11/12) Slide 21 Collaborations • NREL – Dr. Pin-Ching Maness Expressing environmentally- derived hydrogenase in her Synechocystis sp. PCC 6803 system Purified cyanobacterial ferredoxin • Vanderbilt University – Dr. Carl H. Johnson and Dr. Yao Xu Using circadian rhythm modification to enhance expression of O2-tolerant hydrogenases in cyanobacteria Manuscript in preparation Slide 22 Proposed Future Work FY2013 • Continue optimization of promoter strength to achieve maximum expression of active hydrogenase. • Continue to modify small subunit to increase ferredoxin binding. FY2014 • Combine all positive modifications into a single cyanobacterial strain and test for hydrogen production from light and water. Slide 23 Summary • Developed strategies for increasing expression and activity of the environmentally-derived hydrogenase in cyanobacteria Changed the frequency and strength of promoters. Tested a novel T7 polymerase strategy for expression of hydrogenase. Altered the FeS cluster ligation to increase H2 evolution activity • Developed strategies for increasing hydrogenase- ferredoxin interaction Constructed a ferredoxin-hydrogenase fusion protein that maintains activity. Slide 24 .