A Strategy for Design, Redesign, and Optimization of Ethylene Production in E. coli

In collaboration with the University of Colorado-Boulder and LBNL/University of California, Berkeley, we are developing a high-throughput platform approach for prediction and selection to systematically improve isobutanol and ethylene production in E. coli. This project will ultimately combine implementation and testing of predicted modifications based on pathway modeling followed by high-throughput screening of genome-scale (TRMR) and combinatorial pathway (MAGE) libraries to create strains with improved production. Current work at NREL is focused on enzyme and pathway optimization of a chassis ethylene production that will be subsequently utilized for TRMR/MAGE selection methods.

Ethylene is the most highly utilized organic compound used in the production of plastics and chemicals and can be utilized as a precursor for high-energy biofuels. Most ethylene is derived from fossil fuels by steam cracking, resulting in the highest CO₂ emissions in the chemical industry. Ethylene is produced in some bacteria and fungi via an ethylene-forming enzyme (Efe) that uses alpha-ketoglutarate (AKG) and arginine as substrates. Heterologous expression of efe alone is sufficient for ethylene production in a variety of organisms, but the reported productivity is low. In order to improve the often low titers of ethylene produced, an in-depth understanding of optimal expression levels, solubility/stability of Efe, the Efe reaction mechanism, and interactions between the Efe reaction and other metabolic pathways are needed. Our current work is focused on investigation of optimal levels of Efe enzyme for maximal solubility and activity, testing substrate-feeding and/or targeted genetic modifications based on pathway modeling to improve flux to AKG and arginine, and development of high-throughput screening methods to select for increased production of key intermediates and/or ethylene to allow for selection from pooled libraries.