title: A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism.
abstract: BACKGROUND
  FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2.
  
  
  RESULTS
  We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.
  
  
  CONCLUSIONS
  Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.|Wyss Institute, Harvard University, Boston MA 02115, USA.
authors: Buz
authors: B
authors: Barstow
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.
authors: Christina M
authors: CM
authors: Agapakis
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.
authors: Patrick M
authors: PM
authors: Boyle
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.
authors: Gerald
authors: G
authors: Grandl
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.|Wyss Institute, Harvard University, Boston MA 02115, USA.
authors: Pamela A
authors: PA
authors: Silver
authors: Department of Systems Biology, Harvard Medical School, Boston MA 02115, USA.
authors: Edwin H
authors: EH
authors: Wintermute
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