Direct Solar Biohydrogen : Part II Annual Report – June 2007 to May 2008

The overall objective of this research project is to develop a photosynthetic organism for the efficient conversion of sunlight and water into hydrogen fuel. The splitting of water to generate the required electrons and protons, however, also generates molecular oxygen which then poisons the intended catalyst for hydrogen production, an Fe-Fe hydrogenase enzyme. Our main focus, at this time, is therefore the evolution of an oxygen tolerant hydrogenase. In addition, we are studying the requirements for both in vivo and in vitro expression of active hydrogenases. This latter information will be important in moving quickly to engineer the hydrogen producing organism after the required enzyme is developed. Using cell-free synthesis to produce the mutated hydrogenases allows them to be evaluated in a rapid and efficient manner. However, when extensive mutagenesis of a hydrogenase with a relatively simple structure only produced enzymes with more activity but with no improvement in oxygen tolerance, we turned to a hydrogenase with a more complex structure. This, in turn, necessitated significant improvements in the methods used for expression and evaluation of the mutated enzymes. These improvements are now being implemented. For example, reoptimization of the expression gene and the cell-free reagents provided a significant improvement in the cell-free hydrogenase titers. These, in turn, overshadowed a background activity observed in the colorimetric activity assays. We have also discovered two previously unrecognized factors that are required for hydrogenase activation. In addition, significant progress has been made in the development of an ultra-high throughput hydrogenase screening protocol using fluorescently activated cell sorter (FACS) technology. This technology will enable the screening of millions of candidates in the search for oxygen tolerant hydrogenases. Finally, we are making good progress in improving the in vivo expression and maturation of these complicated enzymes in E.coli. This system uses maturation helper proteins from Shewanella oneidensis to help activate hydrogenases from Chlamydomonas reinhardtii and from Clostridium pasteurianum. This feat suggests that this complex expression and maturation system can indeed be moved to different organisms, which bodes well for the future feasibility of engineering the photosynthetic hydrogen producer. Introduction Our overall objective, shown schematically in Figure 1, is to engineer a new microorganism that captures sunlight and channels the energy directly to the formation of hydrogen. As the solar energy is captured by the photolysis center (photosystems I and II), water is split into protons, electrons, and molecular oxygen. The electrons are transferred directly to the electron carrying protein, ferredoxin, which then transfers them to the hydrogenase enzyme that produces the hydrogen. Also illustrated in Figure 1 is the current major challenge for the project. The side product, oxygen, inactivates all known hydrogenases. Thus, the first and most significant challenge for this project is to evolve a natural hydrogenase enzyme to become oxygen tolerant. As will be described, this search has proven to be more difficult than expected. It has been necessary to switch from the evolution of a smaller, simpler hydrogenase to a larger and more complex enzyme with better potential for protecting the oxygen sensitive active site. We are also developing new screening technology using emulsions in which each water droplet is an individual bioreactor to provide dramatically higher throughput for the evaluation of greater numbers of altered hydrogenases. In parallel, we are learning more about the requirements for hydrogenase activation. These insights allow us to improve the cell-free expression system for more effective hydrogenase evolution. They also are guiding the optimization of in vivo hydrogenase expression and maturation to provide information that will help us design the photosynthetic hydrogen producer. Background Strategy for Evolving Hydrogenases. Hydrogenases are very complicated enzymes typically classified as either [NiFe]or [FeFe]-hydrogenases. We have focused on the [FeFe]-hydrogenases as these enzymes provide much faster catalytic rates for hydrogen production. The 3-D structure has been determined for the [FeFe] hydrogenase, CpI, from Clostridium pasteurianum (see Figure 2). Solar Energy