(436a) Comparison of the Isoprenoid Pathways in Marine Diatoms Using Isotope Assisted Metabolic Flux Analysis and Genome Scale Modeling

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.