Metabolic Characterization of Triglyceride-Producing Escherichia coli.
Paul, Julie.
2013
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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 sourc... read moree of oils 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
- Component ID:
- tufts:21980
- To Cite:
- DCA Citation Guide EndNote
