A nitrogen deprivation gradient triggers transcriptional reprogramming for lipid biosynthesis in Auxenochlorella pyrenoidosa.

Nitrogen deprivation is a well-established strategy to enhance lipid accumulation in microalgae, yet the transcriptomic mechanisms underlying a gradient of nitrogen deprivation remain poorly understood. This study investigated the oleaginous microalga Auxenochlorella pyrenoidosa under a sodium nitrate (NaNO₃) gradient (1.5, 1, 0.5, and 0 g L⁻¹). RNA-seq analysis revealed distinct molecular responses tailored to nitrogen availability. Under mild limitation (1 g L⁻¹), nitrate assimilation and nitrogen metabolism were predominantly suppressed. Moderate deprivation (0.5 g L⁻¹) triggered extensive downregulation of the photosynthetic apparatus, encompassing chloroplast function and porphyrin/chlorophyll metabolism. In contrast, severe deprivation (0 g L⁻¹) induced nucleolar stress and upregulated ribosome biogenesis, indicating a fundamental shift in gene expression regulation. Common adaptive mechanisms across all stress levels included the consistent upregulation of the TCA cycle, MAPK signaling, and specific transporters. Furthermore, series cluster analysis identified monotonically expressed genes implicating tRNA processing, the COP9 signalosome, glycolysis, gibberellin response, and lysosomal hydrolases as potential rate-limiting steps governing lipid accumulation. Our findings provide a comprehensive transcriptomic landscape, revealing both tiered and shared regulatory networks, and propose novel targets for engineering lipid production in microalgae under precision nitrogen regulation.