Engineering Escherichia coli to Increase the Production of Free Fatty Acids

Microbial production of free fatty acids (FFAs) and their derivatives from renewable plantderived biomass is considered a promising approach for replacement of petroleum-based production of fuels and chemicals. Microbial production has several advantages, such as sustainable and costeffective production, and direct synthesis of chemicals from renewable. For these reasons, many studies have attempted to redesign microbes through metabolic engineering and synthetic biology for increased production of FFA. One of efficient strategies is to push/pull and block (push: activating first or rate-limiting reaction in a metabolic pathway, pull: activating final reaction, block: inactivating product-degrading). Previously, the genes encoding acetyl-CoA carboxylase and thioesterase were overexpressed to push and pull the carbon flux toward FFA synthesis. Furthermore, genes in the β-oxidation pathway were also deleted to prevent the degradation of FFA. The combined strategy that employed push/pull and block increased FFA production, but the strain exhibited around 0.048 g of FFA/g of glucose, which is equivalent to 14% of the theoretical yield, indicating that further manipulations are required to enhance FFA production. In this thesis, several metabolic strategies were used to construct engineered E. coli based on push/pull and block strategy. First, acetyl-CoA carboxylation bypass was introduced to avoid regulation in the carboxylation. The bypass also provided an alternative route for malonyl-CoA synthesis, altered precursor specificity, and redirected carbon flux from the TCA cycle to FFA synthesis, resulting in 2.5-fold increase in FFA production. Second, mutant thioesterase that is more catalytically active was constructed. The change of amino acid residue in the non-conserved region increased catalytic activity up to two-fold. Employing the mutant thioesterase overcame limited FFA production driven by protein-protein interaction in the fatty acid synthetic pathway, resulting in twofold higher FFA production than employing wild-type thioesterase. Finally, identifying and engineering membrane transport system, indirectly involved in increased FFA production, improved both extracellular and total FFA production. Further engineering including overexpression of transcriptional regulator FadR and phosphoenolpyruvate carboxylase (PPC) increased FFA production. Combinatorial manipulation of the strategies increased FFA production yield (0.24 g of FFA/g of glucose, corresponding to 69% of theoretical yield) which is 5-fold higher than that of the benchmark strain. 6 The works presented in this thesis provide viable strategies for improving production of the FFA, precursor for value-added chemicals such as biodiesel, consumer product (cosmetics, shampoo), and industrial product (lubricants, surface coating). While success was attained in an increase in FFA production titer and yield, the optimized FFA-producing strain could be constructed through further engineering such as increasing NADPH pool, developing active expression system in stationary phase, and reprogramming metabolic pathway.