Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

Ciarán L. Kelly, Constanze Pinske, Bonnie J. Murphy, Alison Parkin, Fraser Armstrong, Tracy Palmer, Frank Sargent (Lead / Corresponding author)

Research output: Contribution to journalArticle

7 Citations (Scopus)
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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 example Escherichia 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 an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.

Original languageEnglish
Pages (from-to)94-104
Number of pages11
JournalBiotechnology Reports
Volume8
Early online date19 Oct 2015
DOIs
Publication statusPublished - Dec 2015

Keywords

  • Bacterial hydrogen metabolism
  • Electron-bifurcation
  • Fermentation
  • Molecular genetics
  • Protein engineering
  • [FeFe]-hydrogenase

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