Unraveling metabolism underpinning biomass composition shift in Scenedesmus obliquus under simulated outdoor conditions using 13C-fluxomics

To render the resulting biomass more attractive and amenable for utilization as the basis for low-carbon intensity bioproducts, single-celled algae need to be biochemically and metabolically poised to assimilate and store the delivered carbon in the fastest and most efficient manner. Accelerating biochemical carbon storage, as primarily carbohydrates or lipids, is critical to achieve the high carbon capture potential that is assigned to algae. To guide strain optimization and engineering for maximizing carbon capture and storage, it is essential to elucidate the link between carbon metabolism and biomass composition. Most published metabolomics work in algae remains largely restricted to ideal and simplified environmental conditions in model organisms, thereby limiting their translation to outdoor implementation. In this work, we utilize ¹³C isotopic labeling to characterize distinct intracellular metabolic fluxes before, during, and after nitrogen depletion-induced compositional shifts in <i>Scenedesmus obliquus</i> UTEX 393. The results indicate that a transition to carbohydrates is characterized by diverting flux to starch instead of replenishing the Calvin cycle for CO₂ fixation whereas the subsequent transition to lipids is fueled by NADPH produced by upregulating the phosphoenolpyruvate carboxylase (PEPC)-malic enzyme (ME) cycle flux. Our work highlights bottlenecks to carbohydrate- and lipid-rich biomass and can guide implementable strategies to control the fate of fixed carbon in <i>S. obliquus</i>.