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

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.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.Nature provides a catalyst for H2 production: hydrogenase metalloenzymes[1]. Hydrogenase active sites are composed only of widely available metals, yet can reduce protons to H2 more rapidly than platinum catalysts[2]. They have therefore been proposed as a substitute for precious metal electrodes for the low-cost, high-volume interconversion of electricity and H2[2,3]. Enzymatic catalysts are particularly well-suited for synthetic integration with specifically biological energy sources. For example, coupling of hydrogenase enzymes with the photosynthetic machinery enables the direct production of H2 from sunlight, as has been demonstrated both in vitro[4] and in vivo[5,6].Several classes of hydrogenases are found in nature[1], distinguished primarily by the metal content of their