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.