283547 Comparison of the Isoprenoid Pathways in Marine Diatoms Using Isotope Assisted Metabolic Flux Analysis and Genome Scale Modeling

Wednesday, October 31, 2012: 8:30 AM
Westmoreland East (Westin )
Andrew Quinn1, Yuting Zheng1, Steven Hutcheson2 and Ganesh Sriram1, (1)Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, (2)Cell Biology and Molecular Genetics, University of Maryland, College Park, MD

Isoprenoids are a diverse class of hydrocarbons formed from five carbon molecules that have high value as therapeutics and biofuel precursors. Large-scale production of isoprenoids through metabolic engineering techniques has received much attention. The production of isoprenoids occurs in two organelles (the plastid and the cytosol) of most photosynthetic organisms. Isoprenoid production in the cytosol is exclusively the result of the mevalonate pathway, while plastidic production occurs via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

We will report the metabolic flux and network analysis toward determining carbon flux through each of these parallel pathways in the model diatom Phaeodactylum tricornutum (Pt). We chose Pt for this study due to its high photosynthetic efficiency and its ability to synthesize large quantities of lipids and other reduced compounds.  Prior isotope labeling studies have shown that the mevalonate and MEP pathways utilize different carbon substrates (Cvejic, J., Rohmer, M., Phytochemistry, 53, 21-28, 2000). We will expand on this work, using isotope-assisted metabolic flux analysis (MFA). This technique is widely used to analyze the labeling patterns of intracellular metabolites from cultures grown on substrates strategically labeled with the 13C isotope of carbon. Knowledge of the carbon rearrangements between metabolites along with the measured 13C enrichment data for each metabolite allows for the calculation of the flux through each reaction. To compare the mevalonate and MEP pathways, which share a number of common metabolites, we will design a set of 13C labeled tracer substrates that create distinct labeling patterns within each pathway. Optimal tracer design is a non-trivial problem, because substrate labels can produce ambiguous labeling patterns, which prevent the determination of certain fluxes. Additionally, we will report the maximum theoretical flux through each pathway by using flux balance analysis employing a model such as those previously reported for Arabidopsis and Zea mays. This computational effort will produce a theoretical flux map showing the fluxes for maximal isoprenoid production, subject to the organism's metabolic constraints.   

We anticipate that this work will provide tools to investigate the relative activity of the two isoprenoid pathways in organisms such as Pt under different conditions. These results, in tandem, will provide the necessary information for any future work focused on rational engineering of Pt to maximize the production of target isoprenoids

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