Hydrogenases, which combine protons and electrons to form hydrogen, are key to the development of biotechnological systems to capture solar energy as hydrogen. Unfortunately, photosynthetic oxygen production and hydrogen production must be separated temporally or spatially due to the sensitivity of the catalytic hydrogenase metal cluster to deactivation by oxygen. The highly active [FeFe] hydrogenases are particularly sensitive. We are using directed evolution to find catalytically improved and oxygen-tolerant [FeFe] hydrogenase mutants superior to the wild-type for industrial applications. We have recently developed an extremely high-throughput screening method based on our ability to activate the complex hydrogenase active site in a cell-free protein synthesis system. Our method uses in vitro compartmentalization, in which emulsification of the cell-free reaction mixture into a continuous oil phase results in isolation of individual mutant genes in femtoliter-volume protein synthesis reactions. FACS then allows candidates to be evaluated at rates of over one million per hour.

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