Biohydrogen production in Escherichia coli – a synthetic biology approach

  • Ciaran L. Kelly

    Student thesis: Doctoral ThesisDoctor of Philosophy


    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 goal a number of alternative synthetic biology approaches were investigated. A synthetic [FeFe]-hydrogenase based on a thermostable NADH-dependent hydrogenase was designed, constructed and expressed in E. coli. 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 cellular metabolism and the resulting strains were characterised using metabolomics and standard microbiological approaches.
    Another approach used in this project involved the construction and expression of active H2-producing synthetic chimeric metalloenzymes, which combined the catalytic activity of two different enzymes: thiosulfate reductase from Salmonella; and E. coli Hyd-2. The activity of this synthetic fusion enzyme could be improved by increasing/maintaining the proton motive force across the cytoplasmic membrane, through the heterologous expression of a proton-pumping proteorhodopsin. Finally the bidirectionality of E. coli Hyd-2, i.e. H2 oxidation and H2 production, was demonstrated, suggesting that it may operate as a quinol release valve in vivo.
    Date of Award2014
    Original languageEnglish
    SupervisorFrank Sargent (Supervisor)

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