Transcriptomic insights into metabolic reprogramming and exo-polysaccharide synthesis in Porphyridium purpureum under gradual nitrogen deprivation

Porphyridium species are known red microalgae for producing valuable bioactive compounds such as sulfated exopolysaccharides (EPS) with diverse industrial biomed-ical applications due to their functional and rheological properties. Recent studies have investigated how abiotic stresses, particularly nitrogen deprivation, affect Porphyridium's metabolic regulation and EPS production through transcriptomic analysis. Still, the mechanisms governing EPS biosynthesis and the involvement of carbohydrate-activated enzymes (CAZymes) remain poorly understood. This study investigated the progressive effects of nitrate consumption on unicellular red alga P. purpureum, by integrating physiological, biochemical, and transcriptomic analyses through RNA-Seq, further validated by RT-qPCR. P. purpureum displayed a gradual, phase-dependent metabolic response to progressive nitrogen stress. EPS release coincided with the decline in nitrate uptake, linking nitrogen availability to carbon redirec-tion towards polysaccharide secretion. Transcriptomic data revealed global metabolic downregulation with targeted upregulation of stress-responsive, carbohydrate catabolic, and nucleotide-sugar synthesis pathways, including upregulation of CAZyme families GT4, GT8, and GT77. Our results give insights into the coordinated nitrogen and carbon metabolic regulation underlying polysaccharide biosynthesis, while open-ing future perspectives on enzyme compartmentalization and regulatory flux distribution under nitrogen stress in P. purpureum. Overall design: RNA-seq profiling of red microalga Porphyridium purpureum cultures exposed to four residual nitrate concentrations (1.5, 0.6, 0.5, 0.3 g·L?¹), with technical triplicates to study the onset of nitrate depletion.