Metabolic Characterization of Triglyceride-Producing Escherichia coli.
Paul, Julie.
2017-04-24T15:11:01.509Z
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Abstract: The goal of this project was to engineer and characterize an Escherichia coli strain that is capable of producing triglycerides. Triglycerides, or oils, are a popular commodity that have various uses, such as the production of biodiesel, lotions, lubricants and cooking oils. They are usually derived from plant sources. This E. coli strain has the potential to become a renewable source of... read moreoils for use in a variety of chemical products. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyl transferase (DGAT). Three liquid chromatography-mass spectrometry (LCMS) methods were used or developed to analyze the reactants (diglycerides and acyl-Coenzyme As (acyl-CoAs)) and product (triglyceride) for the reaction performed by DGAT. A genome-scale model was also used to analyze the potential effects that this inserted pathway may have on metabolism. This was done in an effort to determine the next steps of metabolic engineering in this strain. Results determined that acyl-CoAs were undetected in cell samples. Diglycerides were present at 1.4 mg/(g dcw). Excluding tripalmitin, triglycerides were present at a minimum of 0.5 mg/(g dcw). From the genomic model, E. coli is capable of generating a maximum of 47.6 mmol/(g dcw h) triglycerides from an equivalent flux of diglycerides and acyl-CoAs. Evidence demonstrates that acyl-CoAs are the limiting reactant and can be upregulated with further modifications to the engineered strain. Future experiments will be devoted to increase fatty acid and acyl-CoA concentrations in the engineered triglyceride-producing E. coli.
Thesis (M.S.)--Tufts University, 2013.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisors: Kyongbum Lee, and Blaine Pfeifer.
Committee: Qiaobing Xu.
Keywords: Chemical engineering, and Biochemistry.read less - ID:
- nk322r82s
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