Functional and Structural Analysis of Algal Hydrogenase Combinatorial Mutants (TSE03-D)

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Molecular hydrogen is a promising energy source due to its clean combustion and potential for sustainability. Among hydrogen producing technologies, photosynthetic mechanisms are particularly interesting because of their potentially low energy requirements, as exemplified by the Fe hydrogenase system in the green alga Chlamydomonas reinhardtii. However, hydrogen production in C. reinhardtii is inhibited both transcriptionally and post-translationally by molecular oxygen. These features pose a significant challenge to the development of commercial algal hydrogen generation, which is estimated to require a 70 to 720-fold improvement in oxygen tolerance of the hydrogenase enzyme itself. This project is intended to yield such an enzyme, using the approach of combinatorial mutagenesis (DNA shuffling) to generate mutants that cannot be produced by other methods. Additional objectives are to develop a novel colorimetric assay for hydrogen production into an automated, high-throughput screen to evaluate mutant libraries for optimal diversity before undertaking in-depth analyses and to investigate a promising new method known as Random Chimeragenesis on Transient Templates (RACHITT) that may generate even more useful libraries. Most-improved genes will be recovered and re-shuffled as necessary, and functional and structural modeling studies will be conducted on the best mutants to reveal mechanisms of improvement. The intended product is an oxygen-tolerant Fe hydrogenase that will ultimately facilitate commercial algal hydrogen production.