BioHydrogen Production in E. coli – A Synthetic Biology Approach

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Ciaran Kelly, Alison Parkin, Tracy Palmer, Frank Sargent

University of Dundee, United States

In the 21st Century molecular hydrogen (H2) has become an essential industrial commodity. It is widely heralded as an exciting alternative to petroleum-based transportation fuels and also plays indispensable roles in many other important industrial processes, including hydrogenation of fats and oils, methanol production, and ammonia production – an essential component of agricultural fertilizers. Biohydrogen (Bio-H2) is molecular hydrogen produced by microorganisms and is an exciting prospect as a fully renewable, commercially-viable second-generation biofuel. Bio-H2 can be produced at ambient temperatures by metal-dependent hydrogenase enzymes, with potentially no CO or H2S contamination, and is a carbon neutral/positive process. Escherichia coli naturally produces Bio-H2 during mixed-acid fermentation via its own endogenous nickel-dependent hydrogenase enzymes. The main aim of this project is to enhance Bio-H2 production by E. coli and to achieve this synthetic hydrogenases have been designed, constructed and expressed. The structure and activity of the synthetic hydrogenase has been characterised in vitro using a number of techniques including autoradiography, spectroscopy, SEC-MALLS, protein-film voltammetry and H2 production assays. Metabolic engineering of various E. coli strains and directed protein evolution has been carried out to integrate this synthetic hydrogenase activity into cell metabolism and the resulting strains were characterised using metabolomics and standard microbiological approaches. Finally, a number of alternative synthetic biology approaches to increase H2 production E. coli are currently being investigated, including the construction and expression of unusal enzyme chimeras.