Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Synechocystis PCC 6803 Strains for Maximal, 24-Hour Production of Hydrogen Gas

This project is using the cyanobacterial species Synechocystis PCC 6803 as a model to pursue two parallel lines of inquiry initially, with each line addressing one of the two main factors affecting H2 production in PCC 6803: NADPH availability and O2 sensitivity. H2 production in PCC 6803 requires a very high NADPH:NADP+ ratio, that is, that the NADP pool be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-H2ase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations, reportedly due to binding of O2 to the active-site. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild-type (WT) PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we are using H2 production profiling and metabolic flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work will provide rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we are using site-directed and random mutagenesis to create mutants with H2ase enzymes exhibiting greater O2 tolerance (and perhaps higher H2 production activity). The objectives of the research are addressed via the following four tasks: