(326f) A High-Throughput in Vitro Photosynthetic Hydrogen Production Screen

Coupling photosynthetic water-splitting with the production of hydrogen (H₂) is an attractive method to produce this important commodity chemical and potential, next-generation fuel. Photobiological H₂ production requires integration of hydrogen-producing enzymes, hydrogenases, into native photosynthetic electron transport chains. However, heterologous hydrogenases are unstable in the presence of oxygen, an unavoidable byproduct of photosynthesis, and inefficiently couple with the native photosynthetic machinery of organisms like cyanobacteria. Engineering hydrogenases for increased H₂ production is limited in part by a lack of robust, high-throughput screening methods. In vitro expression and screening of enzyme mutants would enable more rapid testing of larger library sizes than in vivo systems, in part due to challenges in genetic engineering of photosynthetic organisms.
         Here, we will discuss the development and optimization of an in vitro photosynthetic H₂-production pathway using photosystems isolated from a cyanobacterium. Additionally we will describe the use of a high-throughput assay for assessment of H₂ production rates of hydrogenases coupled to the isolated photosystems in microtiter plates. This approach can be extended to the high-throughput screening of in vitro synthesized enzyme libraries for increased photobiological H₂ production rates. Additionally, this work will provide a powerful new assay to interrogate photosynthesis in vitro allowing fine-tuned control over the reaction environment by modulating enzyme and reagent concentrations.