The TCA cycle and pentose phosphate pathway are linked to lipid droplet expansion in nitrogen-starved Lipomyces starkeyi cells

In eukaryotic cells, lipid droplets (LDs) play critical roles in storing energy, preventing lipotoxicity, promoting membrane biogenesis, and regulating stress responses, contributing to the maintenance of cellular homeostasis. Similar to white adipocytes under overnutrition, the oleaginous yeast Lipomyces starkeyi cells form a single giant LD during nitrogen deprivation. Under the same conditions, mitochondria form elongated tubules and sheets in a close proximity to a giant LD, although the significance of this mitochondria-LD proximity remains unclear. Here, we show that inhibition of fatty acid synthesis leads to strong suppression of LD expansion in nitrogen-starved L. starkeyi cells. Metabolomics analysis reveals that the TCA cycle intermediates including citric acid, a key precursor for fatty acid synthesis, decrease in cells undergoing LD expansion. In contrast, the pentose phosphate pathway intermediates increase in a manner dependent on fatty acid synthesis. Inhibition of the pentose phosphate pathway, which generates NADPH, a key electron donor for fatty acid synthesis, strongly suppressed LD expansion. Surprisingly, nitrogen-starved L. starkeyi cells also accumulate carnitine, a critical carrier that mediates transport of fatty acids to mitochondria and accelerates beta-oxidation for energy production. Our findings raise the possibility that, under nitrogen starvation, L. starkeyi cells activate fatty acid synthesis with citric acid and NADPH from the TCA cycle and the pentose phosphate pathway, respectively, thereby facilitating energy production and storage concurrently via the mitochondria-LD proximity.