P129: Engineering short-chain fatty acid biosynthesis using thioesterase substrate specificity

Biological synthesis of short-chain fatty acids and alcohols is of interest for production of biofuels, industrial compounds, and pharmaceuticals from renewable resources.  To expand the range of fatty acids and alcohols produced by Escherichia coli, the Prather lab has implemented a coenzyme A (CoA)-dependent pathway that can ligate acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs.  In an effort to increase titers of desired products and reduce early pathway termination products, we have focused on understanding and improving the specificity of the thioesterase enzyme, which is responsible for releasing the fatty acid from CoA.  A total of 62 thioesterases from diverse bacteria were inserted into a pathway that begins with condensation of acetyl-CoA to a molecule of the acyl-CoA derivative of exogenously provided propionic, butyric or isobutyric acid. The culture supernatants of E. coli MG1655 expressing the genes for this pathway with each thioesterase were analyzed via high performance liquid chromatography to determine the titers of desired short-chain fatty acids. Functional screening revealed thioesterases that yield greater titers of saturated, unsaturated and 3-hydroxy short-chain fatty acids of interest such as valerate, trans-2-pentenoate, and 3-hydroxy valerate.  To determine if the increased product titers are the result of shifts in thioesterase substrate specificity, in vitro biochemical analysis is being performed on active thioesterases.  This work will provide greater control of thioesterase specificity, thereby expanding the diversity of fatty acid and alcohol products from CoA-dependent biosynthesis and increasing titers of current products.