Graduate Thesis Or Dissertation
 

Metabolic engineering of cyanobacteria to optimize photobiological hydrogen production

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/5x21tj26z

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  • Photobiological hydrogen production -- the use of photosynthesis to split water into hydrogen and oxygen -- presents an opportunity for efficient and large-scale solar biofuel development. One of the major challenges in this area is competition for reductant between the hydrogen-evolving hydrogenase enzyme and other metabolic pathways. In this work we first develop and characterize a family of inducible promoters allowing for nitrogen-regulated gene expression in cyanobacteria. These four promoters are all induced by nitrate and repressed by ammonium ion but differ in maximal activity and dynamic range. We then apply these promoters to create Synechocystis sp. PCC 6803 strains in which two major competing pathways -- cyclic electron flow around photosystem I and carbon assimilation -- are inducibly downregulated in the presence and absence of the Mehler reaction, another competing pathway. These strains produce hydrogen continuously in the light when grown in ammonium but not when grown in nitrate, demonstrating in vivo function of the nitrogen-regulated metabolic switch. Finally, we examine hydrogen production and chlorophyll fluorescence from these strains in the presence of multiple oxygen levels, oxygen scavengers, and chemical inhibitors to identify further metabolic pathways that may inhibit hydrogen production. These pathways are logical targets for genetic manipulation with the goal of optimizing electron flux from water to hydrogen. The inducible promoters developed here will be applicable to a wide range of cyanobacterial biotechnology, and implementation of a metabolic switch between growth-optimized and hydrogen-optimized phenotypes represents a first step towards development of a commercially viable biohydrogen strain.
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