Production of Ethylene Production from the Common Metabolite, 2-Oxoglutarate, by the Ethylene-Forming Enzyme (EFE)

This project aims at an atomistic description of the so-called 'ethylene-forming enzyme' (EFE), which catalyzes a unique reaction, wherein the common metabolite, 2-oxoglutarate (2OG), is converted to the important commodity chemical, ethylene. EFE is being vigorously leveraged within the biotech industry for novel, solar-driven, carbon-neutral bioprocesses to produce ethylene, one of the most important building blocks of the chemical industry. Insight obtained from the proposed studies could guide the rational design of microbial bioprocesses employing EFE and could therefore impact the world's energy infrastructure and economy.Ethylene is one of the most versatile and broadly used building blocks in polymerization reactions carried out in the chemical industry. It is used for manufacturing of textiles, plastics, solvents and gasoline in the C5-C10 range. Currently, ethylene is produced by steam cracking of fossil fuels in a process that requires vast amounts of energy and has a negative carbon footprint. Therefore, alternative approaches for production of ethylene are being vigorously pursued. An attractive alternative approach for ethylene production involves biotechnology, specifically the use of genetically engineered microorganisms in which the biosynthetic path for ethylene production is imported. The so-called ethylene-forming enzyme (EFE) is an attractive target for these endeavors because it catalyzes the conversion of the common metabolite of the tricarboxylic acid cycle, 2-oxo-glutarate (2OG), to ethylene. EFE belongs to the large group of Fe(II)- and 2OG-dependent oxygenases that couple the decarboxylation of 2OG to succinate and CO2 to the oxidation of their prime substrates, but it is unique in the sense that it is the only Fe/2OG oxygenase which can convert 2OG to ethylene.During the previous funding cycle, we dissected the mechanism of the unusual reaction catalyzed by EFE. Our experimental evidence reveals a hybrid radical-polar mechanism with a second, previously unrecognized branchpoint. The key propionate-3-carbonate intermediate can break down either to ethylene and CO2 or to 3-hydroxypropionate (3HP). 3HP is also an important monomer for the polymer industry. Our observation that EFE can be enticed to generate 3HP from the common metabolite, 2OG, affords the opportunity for synergistic engineering of bioprocesses for both 3HP and ethylene. We will use a combination of biochemical, kinetic, spectroscopic, and crystallographic approaches to provide additional knowledge of how EFE controls its reactivity. These experiments could provide important insight for the design of microorganisms for the production of important commodity chemicals.