Metabolic Flux Analysis for Metabolic Engineering of Marine Organisms

Abstract

We explored the metabolic pathways in two industrially relevant marine microorganisms to understand their core metabolic capabilities. It is necessary to track how an organism distributes organic building blocks throughout its metabolic pathways so that we can devise strategies to alter its metabolism and reroute substantial metabolic flux towards target compound(s). Though we cannot measure intracellular metabolic fluxes directly, we can retro-biosynthetically calculate them by supplying substrates labeled with non-radioactive isotopes to an organism. We then measure the resulting isotope labeling patterns of metabolites and calculate the fluxes that produced them.

We addressed three goals with our research, (i) resolving questions surrounding organic carbon metabolism in the diatom Phaeodactylum tricornutum (P. tricornutum), (ii) identifying reactions in a putative photosynthetic carbon concentrating mechanism in P. tricornutum and (iii) mapping central carbon metabolism of the cellulolytic aerobe Saccharophagus degradans (S. degradans).

Towards goal (i) we show that P. tricornutum predominantly consumes glucose, as opposed to atmospheric CO2, under mixotrophic conditions using the Entner-Doudoroff (ED) glycolytic pathway instead of the more common Embden-Meyerhof-Parnas pathway (EMP). We utilized metabolic flux analysis (MFA) to discover that acetate is metabolized for energy production instead of for biomass formation during mixotrophic growth on CO2 and acetate. Finally, we developed a method for measuring isotopic labeling in polyunsaturated fatty acids via gas chromatography-mass spectrometry (GC-MS), and demonstrated its utility in resolving outstanding questions about glucose metabolism by P. tricornutum. Towards goal (ii) we utilized isotope labeling and gene silencing in combination to identify pyruvate carboxylase as a key enzyme in a C4 carbon concentrating mechanism in P. tricornutum, while also ruling out phosphoenolpyruvate carboxylase as a key enzyme in the pathway. Towards goal (iii) we present 13C-MFA of aerobic consumption of glucose, xylose, and cellobiose by S. degradans. This is the first reported MFA of cellobiose metabolism and one of only a handful analyzing xylose metabolism in an aerobic microorganism.