(474b) Engineering Production of Fatty Acid Derived Products in Escherichia Coli through Functional Genomics and High Throughput Screening

Biological hydrocarbons offer many advantages over cellulosic ethanol including higher energy density, lower water solubility, and compatibility with current engines. Biodiesel, the methyl or ethyl esters of fatty acids, is currently the second leading biofuel to ethanol. Similar to corn-based ethanol, the production of biodiesel is limited by the availability of oil crops. Metabolic engineering offers the tools to link cellulosic technologies with the production of more desirable fuels. One potential platform for the synthesis of diesel-like hydrocarbons, is the production of lipids in bacteria such as Escherichia coli for subsequent reduction to hydrocarbon fuels. We have successfully engineered E. coli to overproduce C12 fatty acids by overexpressing a plant thioesterase in a β-oxidation mutant, Lennen et al., Biotechnology and Bioengineering, 2010. In these initial studies, we identified the thioesterase catalyzed cleavage of acyl chains from the corresponding aycl-ACP as the key step in the biosynthesis of free fatty acids. If the gene encoding the thioesterase is overexpressed beyond an optimal level, we observe a dramatic decrease in cellular fitness and fatty acid titer. Here we present recent progress using functional genomics and high throughput screening to elucidate the molecular link between thioesterase overexpression and reduced fitness.