A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

BACKGROUND: FeFe-hydrogenases are the most active class of H(2)-producing enzymes known in nature and may have important applications in clean H(2 )energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O(2). 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 O(2 )levels eliminate growth. This pathway forms the basis for a genetic selection for O(2 )tolerance. Genetically selected hydrogenases did not show improved stability in O(2 )and in many cases had lost H(2 )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 H(2 )metabolism. Our results also indicate a H(2)-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H(2)-activating catalysts.