Increasing Hydrogen Photoproduction By Genetic Engineering

Reversible hydrogenases are enzymes which can reversibly produce molecular hydrogen from hydrogen ions. The overall objective of this project is to genetically transform certain high hydrogen producing strains of cyanobacteria with cloned reversible hydrogenase genes derived from well characterized cyanobacterial species. Within the past few years, several reversible hydrogenase gene clusters have been sequenced (Anacystis nidulans, Synechocystis PCC 6803, and Anabaena variabilis), and these sequences are available from GenBank. Based on the sequences, suitable PCR primers will be synthesized and used to amplify them. These will then be cloned into E. coli with the use of a commercially available cloning vector. Upon the successful cloning of the reversible hydrogenase gene clusters, these will be placed in a shuttle vector which can replicate in both E. coli and a given strain of cyanobacteria. The shuttle vector containing the reversible hydrogenase gene cluster will be introduced back into the cyanobact rial strain from which the cluster originated in the anticipation that the resulting higher copy number of the reversible hydrogenase genes will cause an increase in the reversible hydrogenase protein and in turn an increase in hydrogen production. The hydrogen production of the transformed cyanobacterial strain will be measured by gas chromatography. In addition, the amplified reversible hydrogenase gene cluster of a given strain will be introduced into the other cyanobacterial strains which contain a known cluster, and their hydrogen production measured. This will establish whether the reversible hydrogenase gene cluster of a particular strain can augment and complement the function of the gene cluster of a different strain in such a way as to increase the production of hydrogen. Finally, the cloned reversible hydrogenase gene clusters will be introduced into certain strains of nitrogen fixing cyanobacteria which already have a high rate of hydrogen production from their nitrogenase enzyme. The hydrogen production of the genetically transformed cyanobacteria will be measured by gas chromatography in order to determine if the reversible hydrogenase can complement and supplement the function of nitrogenase in a manner which increases the production of hydrogen