290176 Optimizing Growth Conditions of Escherichia Coli Through Metabolic Flux Analysis to Enhance Fatty Acid Production

Monday, October 29, 2012
Hall B (Convention Center )
Jordan Baker, Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD

Of all of the petroleum used in the current society, the majority of it goes to making gasoline and fuels. A much lower percentage of the petroleum goes to making petrochemicals, which have equivalent total revenues. A smaller volume of the petroleum is used for petrochemicals, but they are just as lucrative. One way to decrease the cost of petrochemicals is to synthesize precursors for petrochemicals using renewable, biological sources.

           One fast way to produce fatty acids, a necessary precursor for petrochemicals, is to use Escherichia coli, which naturally produce medium length (C14 and C16) fatty acids. E. coli is an ideal organism to choose because they grow fast and their genome is well modeled. Because the genome is well modeled, we can alter the DNA of the E. coli to try to increase fatty acid production.

            E. coli produces approximately 5 grams of fatty acids per liter in the LB rich media containing proteins, amino acids, and other carbon sources, whereas it only produces around 1.7 grams of fatty acids per liter in minimal M9 media with only glucose as a carbon source. The concentrations of the other carbon sources and ingredients in the rich LB media are unknown, so I wanted to optimize the growing conditions in M9 minimal media for a previously modified E. coli strain by using metabolic flux analysis to model the metabolism of the E. coli. The modified strain I was working with was ML103 E. coli, which is wild type E. coli with a FadD (fatty acid oxidation) knockout and the pxz18Z plasmid, which contains FabZ (dehydration of fatty acid in elongation cycle) overexpression and Acyl-ACP (termination of fatty acid elongation by hydrolyzing an acyl group on fatty acid chain) overexpression. While researching what differences could account for the increased fatty acid production, I hypothesized that amino acid additions or a different carbon to nitrogen ratio in the LB media accounts for the increased fatty acid production.

            The first step in my project tested the effect of aeration space on the growth and fatty acid production of the E. coli. I grew E. coli in three different containers: 250mL glass flask, 20mL glass test tube, and 15mL plastic test tube. I measured the growth, optical density (spectrophotometer), and fatty acid production (gas chromatography – flame ionization detector) of all the samples at varying time points throughout the growth.

I found that the samples with the largest aeration space had the highest fatty acid production. After analyzing the data, I deduced, from the growth curve, that up to 24 hours, the cells are still in log phase, and the glucose is used for cell proliferation and growth. After 24 hours, the carbon in the glucose goes to making fatty acids, which is limited by the amount of oxygen since fatty acid production uses oxygen. The largest aeration space had the highest concentration of oxygen available for absorption for respiration to provide ATP, which is necessary for FA biosynthesis.

            My second project was to test whether the addition of amino acids to the media would increase fatty acid production. I added varying concentrations of all twenty common amino acids and grew the E. coli. I measured the optical density (spectrophotometer), glucose uptake (high-pressure liquid chromatography), acetate production (HPLC), and fatty acid production (FC-FID) at varying time points throughout growth.

            I found that the cells with the highest concentration of amino acids grew faster, but had a lower fatty acid production. These cells also had the highest acetate production. From these results, I concluded that up to 24 hours, the cells utilized the amino acids from the media to grow and divide, which is why they grew at a faster rate. In order to utilize the amino acids, the E. coli used an ABC transport system, which is known from literature, which requires two moles of ATP per mole of substrate transported. After 24 hours, the carbon from the glucose went to make acetate in the E. coli with higher concentrations of amino acids in the media. Because the cells used ATP to utilize the amino acids, the cells had a decreased intracellular concentration of ATP. Fatty acid biosythensis requires ATP, but with a decreased ATP concentration, the cells produced acetate instead because acetate production produces ATP. These results show that amino acid supplements decreased fatty acid production.

            My final experiment tested whether altering the carbon to nitrogen ratio would increase fatty acid production. I added varying amounts of ammonium chloride, the usual source of nitrogen in M9 media, to the media. I increased (nitrogen rich) and decreased (nitrogen limited) the nitrogen levels compared to the control M9. I then measured optical density (spectrophotometer), glucose uptake (HPLC), acetate production (HPLC), and fatty acid production (GC-FID) at various time points in the growth.

            The results showed that the control M9 had the highest fatty acid production and any alterations to the carbon to nitrogen ratio decreased fatty acid production. Nitrogen is used for making amino acids and proteins by the E. coli and transported into the cells by one of two pathways. In the nitrogen limited conditions, two enzymes work to bring nitrogen into the cell by hydrolyzing ATP (glutamine synthetase and glutamate synthase). In nitrogen rich conditions, glutamate dehydrogenase uses NADPH as an energy source to import nitrogen. In nitrogen-limited conditions, the cells have a decreased level of ATP, which in turn decreases fatty acid biosynthesis, since that requires ATP. In nitrogen rich conditions, the cells have a decreased level of NADPH and a decreased fatty acid production because the NADPH is also an important energy source in fatty acid biosynthesis. These enzymes are also well known from literature. The conclusion is that the M9 has the optimal nitrogen concentration to utilize both pathways to not decrease ATP or NADPH too much since fatty acid biosynthesis needs both molecules to produce fatty acids.

            At the end of the internship, no significant media component to increase fatty acid production was found, but there was a gain in knowledge about E. coli, the media, and the transport systems.

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