(485g) Production of Photosystem I/ Hydrogenase Fusion Proteins for Improved Electron Transport in Photo-Induced Hydrogen Production

Photobiological hydrogen production using the energy of sunlight has the ability to provide a sustainable means of producing a practical and efficient fuel to replace current petroleum-derived fuels. The photosystem I (PSI)-mediated photoproduction of hydrogen has been demonstrated with reactions centers isolated from plants (such as spinach leaves) and microorganisms (such as cyanobacteria). When the source of PSI is a thermophillic microorganism (for example, the cyanobacterium Thermosynechococcus elongatus), the hydrogen production system exhibits increased thermal stability with hydrogen evolution rates increasing up to 55⁰C. This system is also temporally stable and displays no measurable change in hydrogen evolution rate for periods exceeding 80 days.

In previous in vitro photobiological hydrogen production systems, platinum metal was used to catalyze the formation of hydrogen at the stromal end of PSI. However, hydrogenase enzymes can catalyze the same reaction and could potentially displace the requirement of the precious metal platinum. While it is possible to use aqueous in vitro mixtures of PSI and hydrogenase, the formation of direct fusion proteins of hydrogenase and PSI have demonstrated five times the hydrogen evolution rate of mixtures of the native complexes. Therefore, we seek to replace the platinum catalyst in our current construct with an oxygen tolerant Ni-Fe hydrogenase and to form a hybrid protein by engineering a gene to express a fusion of a hydrogenase from Ralstonia eutropha H16 and the stromal-exposed subunit PsaE of PSI from T. elongatus. A PsaE-free mutant of PSI will simultaneously be formed by genetically disrupting the expression of the PsaE subunit of a native PSI; this will allow in vitro reconstitution of the desired PsaE-hydrogenase fusion protein with PsaE-free PSI. We present the results of our efforts to clone the knockout gene for PsaE, which is used to generate the PsaE deficient PSI with T. elongatus as the expression host. We also discuss the isolation of the megaplasmid DNA from R. eutropha H16 and the amplification and subcloning of the HoxKG gene (which codes for the membrane-bound Ni-Fe hydrogenase) into an appropriate vector. Finally, we report the results of the formation of the final fusion protein.