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

<p>Abstract</p> <p>Background</p> <p>FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.</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 O₂levels eliminate growth. This pathway forms the basis for a genetic selection for O₂tolerance. Genetically selected hydrogenases did not show improved stability in O₂and in many cases had lost H₂production 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 H₂metabolism. 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.</p

Network-Scale Engineering: Systems Approaches to Synthetic Biology

The field of Synthetic Biology seeks to develop engineering principles for biological systems. Modular biological parts are repurposed and recombined to develop new synthetic biological devices with novel functions. The proper functioning of these devices is dependent on the cellular context provided by the host organism, and the interaction of these devices with host systems. The field of Systems Biology seeks to measure and model the properties of biological phenomena at the network scale. We present the application of systems biology approaches to synthetic biology, with particular emphasis on understanding and remodeling metabolic networks. Chapter 2 demonstrates the use of a Flux Balance Analysis model of the Saccharomyces cerevisiae metabolic network to identify and construct strains of S. cerevisiae that produced increased amounts of formic acid. Chapter 3 describes the development of synthetic metabolic pathways in Escherichia coli for the production of hydrogen, and a directed evolution strategy for hydrogenase enzyme improvement. Chapter 4 introduces the use of metabolomic profiling to investigate the role of circadian regulation in the metabolic network of the photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942. Together, this work demonstrates the utility of network-scale approaches to understanding biological systems, and presents novel strategies for engineering metabolism