(169g) Protein Engineering for Enhanced Photo-Production of Hydrogen
AIChE Annual Meeting
2010
2010 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Protein Engineering III - Applications
Monday, November 8, 2010 - 5:25pm to 5:45pm
Provision of an abundant, clean and secure sustainable energy source is one of the key energy challenges facing mankind. New approaches towards clean and sustainable energy are currently underway and sustainably produced hydrogen is considered to be a highly efficient energy source. It has been known since the turn of the century that microorganisms have the capability to produce hydrogen. The metabolism of hydrogen in photosynthetic microorganisms such as algae has been studied since the 1930s and early 1940s.
In previous in vitro photobiological hydrogen production systems, platinum metal was used to catalyze the formation of hydrogen at the stromal end of Photosystem I (PSI) reaction center from a thermophillic cyanobacterium, Thermosynechococcus elonagatus. 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. The psaE gene from T. elongatus will be optimized for improved expression in R. eutropha. 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.