Metabolic Characterization of Triglyceride-Producing Escherichia coli.
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 sour... read morece 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.
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