(798h) Isotope Labeling Experiments Enable Dissection of Parallel Carbon-Reducing Pathways in the Marine Diatom Phaeodactylum Tricornutum

In algae and plants, the 2-C-methyl-D-erythritol phosphate (MEP) and mevalonate (MVA) pathways independently produce isopentenyl pyrophosphate (IPP), an important precursor to a spectrum of highly reduced molecules that are biofuel and biochemical precursors. For example, numerous photosynthetic species convert IPP into the branched C₅ alkene isoprene. Once extracted from a cell, isoprene can be polymerized into true diesel fuel or natural rubber. Therefore, an organism engineered to produce high quantities of IPP could serve as a robust bio-factory for highly reduced fuels far superior to the current generation of bio-alcohols and biodiesel.  

Photosynthetic organisms offer a significant advantage over heterotrophic bacteria and yeast due to their ability to grow on atmospheric CO₂ in addition to reduced carbon substrates. This eliminates the necessity of costly sugars derived from terrestrial crops or cellulosic biomass. One promising organism is the unicellular marine diatom Phaeodactylum tricornutum (Pt). Pt is highly prized for its ability to efficiently sequester carbon dioxide using a unique C4 pathway, as well as for its propensity to convert a large proportion of its biomass into reduced lipids. 

Like many photosynthetic organisms, Pt operates a plastid-localized MEP pathway and a cytosolic MVA pathway. Using isotope labeling techniques, researchers have previously shown that the organism utilizes the MEP pathway to synthesize phytol, a precursor to photosynthetic pigments, whereas the MVA pathway synthesizes steroids such as brassicasterol. The plastidic and cytosolic pools of the IPP precursor are considered separate from one another; however, investigations with Arabidopsis thaliana and Nicotiana tabacum have hinted at some degree of mixing between the two compartments in those organisms. Any effort to overproduce IPP in Pt will require an understanding of the degree of mixing between these two pathways so that the metabolic pathways can be rewired to both maximize the flow of carbon towards a large pool of available IPP, while also synthesizing sufficient steroids and carotenoids to maintain high cell viability and growth.

In this presentation, we will report the dissection of the metabolic cross-talk between the compartmentalized MEP and MVA pathways in Pt based upon in vivo experiments employing enzyme inhibition and isotope-assisted metabolic flux analysis.  By inhibiting one pathway and measuring the abundances of key downstream metabolites, we will determine whether the organism is able to reroute carbon through the uninhibited isoprenoid pathway and across the plastid-cytosol membrane. We will also feed various carbon substrates strategically labeled with the ¹³C isotope of carbon. By measuring the labeling patterns of intracellular metabolites, we will construct metabolic flux maps highlighting how the organism alters its’ central carbon metabolism to compensate for the inhibition of the isoprenoid pathways. We anticipate that these results will reveal efficient and effective strategies to increase IPP production in Pt whilst also maintaining adequate levels of essential, secondary isoprenoids.