| Summary: | <p>Abstract</p> <p>Background</p> <p>FeFe-hydrogenases are the most active class of H<sub>2</sub>-producing enzymes known in nature and may have important applications in clean H<sub>2 </sub>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<sub>2</sub>.</p> <p>Results</p> <p>We have developed a synthetic metabolic pathway in <it>E. coli </it>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<sub>2 </sub>levels eliminate growth. This pathway forms the basis for a genetic selection for O<sub>2 </sub>tolerance. Genetically selected hydrogenases did not show improved stability in O<sub>2 </sub>and in many cases had lost H<sub>2 </sub>production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.</p> <p>Conclusions</p> <p>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 <it>in vivo </it>assays to aid in the engineering of synthetic H<sub>2 </sub>metabolism. Our results also indicate a H<sub>2</sub>-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H<sub>2</sub>-activating catalysts.</p>
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