| Petroleum derived products are in high demand that is predicted to exceed supplies in the future. Therefore, renewable alternatives for fuels and petrochemicals must be developed to avoid geopolitical, economic, and environmental disasters. Microbes, which produce long, hydrocarbon compounds (acyl chains) in the synthesis of the cellular membrane, are an attractive catalyst for production of these compounds. In this thesis, metabolic engineering strategies were applied to Escherichia coli to produce free fatty acids (FFAs) as precursors to a wide range of fuels and chemicals. Reactor and media conditions were studied to indentify optimum regimes for FFA production. To that end, an antibiotic free, FFA overproducing strain of E. coli was constructed and cultivated in continuous culture to generate parameters for a kinetic model of FFA production. Additionally, cells starved for phosphate were shown to be capable of prolonged FFA production in stationary phase with productivities three fold higher than under carbon limitation. Using these strategies, yield surpassed 40% of maximum theoretical values.;In addition to fuels, FFA can be converted to fatty alcohols, which sell for 2.5 times the price of diesel, and represent a 3 billion dollar market. Prior efforts to produce fatty alcohols via FFA biosynthesis failed to reach 10% of theoretical maximum yield. In this thesis, balanced expression of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase resulted in increased production of fatty alcohols. Applying optimal reactor and media conditions from FFA overproduction, fatty alcohol yields of over 40% of theoretical maximum were achieved.;Overall, this thesis demonstrates an effective strategy for finding production regimes that optimize FFA productivity and yield. However, while the yield increases are a significant step, further improvement is necessary to achieve economic viability. Genetic modifications to regulatory or alternative pathways will be required to increase carbon flux to FFA derived products. Additionally, understanding the effect of fatty alcohol production on cell viability could provide insight into methods of increasing production. Thus, while we have more than doubled previous fatty alcohol yield values, the aforementioned strategies should provide insight into reaching the maximum theoretical yield. |
