(627d) Insight into Lipid Biogenesis Using Stable Isotope Tracers Coupled with NMR Spectroscopy and Mass Spectrometry

Algae have great potential for use in biofuel and specialty chemical production due to inherit growth advantages, high triacylglyceride (TAG) content per cell, and the use of renewable energy sources (i.e., sunlight) along with the potential to balance atmospheric carbon levels.  However, there is a lack of basic algal research in lipid biogenesis and metabolism that would aid industry and advance the development curve.  Algal TAG accumulation is the net result of TAG synthesis minus utilization and typically occurs when cellular cycling is stunted or arrested due to nutrient limitation, environmental stress, or by chemical addition.  Here we report on a simplified metabolomics method using ¹H and ¹³C NMR to directly monitor ¹³C incorporation and recycling in real-time, using live cells, during bicarbonate enhanced TAG accumulation induced by N-deplete culturing.  Results indicate rapid carbon incorporation into sucrose (within 15 min) directly after N-depletion, which increased in cellular concentration for 16 hours after which a steady-state concentration was observed.  Within four hours, TAG accumulation was observed.  After 48 hours 65-75% of the carbon incorporated into fully saturated or mono-unsaturated fatty acids was found to be derived from extracellular dissolved inorganic carbon.  Conversely, the majority of poly unsaturated and omega-3 fatty acids that accumulated post N-depletion were recycled presumably from preexisting cellular membranes (biomass synthesized prior to N-depletion).  The extent of ¹³C incorporation into fatty acids, from either extracellular dissolved inorganic carbon, or from recycled intracellular biomass, was confirmed using gas chromatography mass spectroscopy.  In conclusion, this work highlights state of the art metabolomics techniques that expand on current metabolomics methods by avoiding cellular extraction while reducing the amount of untargeted metabolite detection and identification. This study answers key lipid biogenesis questions such as the extent to which de novo fatty acid synthesis versus recycled fatty acids takes place.