title: Integration of an [FeFe]-hydrogenase into the anaerobic metabolism ofEscherichia coli.
abstract: Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for exampleEscherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in anE. colihost directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase fromCaldanaerobacter subterranus.The synthetic operon was stably integrated into theE. colichromosome and shown to produce an active hydrogenase, however no H2production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin fromThermotoga maritimawas found to be essential to drive H2production by this system. This work provides genetic evidence that theCa.subterranus[FeFe]-hydrogenase could be operatingin vivoas an electron-confurcating enzyme.
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Ciarán L
authors.initials: CL
authors.lastname: Kelly
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Constanze
authors.initials: C
authors.lastname: Pinske
authors.affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, UK.
authors.forename: Bonnie J
authors.initials: BJ
authors.lastname: Murphy
authors.affiliation: Department of Chemistry, University of York, Heslington, York YO10 5DD, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Parkin
authors.affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, UK.
authors.forename: Fraser
authors.initials: F
authors.lastname: Armstrong
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
keywords: Bacterial hydrogen metabolism; Electron-bifurcation; Fermentation; Molecular genetics; Protein engineering; [FeFe]-hydrogenase
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