DUR3 as a Molecular Lever for Coordinated Nitrogen and Phosphorus Uptake in Microalgae

Nitrogen (N) and phosphorus (P) are essential macronutrients for plant growth and major pollutants driving aquatic eutrophication. Microalgae represent a sustainable biological platform for nutrient recovery and circular utilization from wastewater; however, the molecular mechanisms governing efficient urea assimilation and its coordination with phosphorus uptake remain inadequately characterized. This study investigated how overexpression of the high-affinity urea transporter gene DUR3 enhances nutrient scavenging capacity in the model green alga Chlamydomonas reinhardtii. The DUR3-overexpressing line exhibited concentration-dependent growth responses to urea, showing significant promotion at low-to-moderate levels but inhibition at high urea concentration or under pure-urea conditions, where DUR3-overexpressing (DUR3-OE) was more severely inhibited than the wild-type (WT). Notably, the DUR3-OE consistently increased chlorophyll content and photosynthetic efficiency (Fv/Fm) under ammonium, urea, and mixed-N regimes. Under low-urea conditions, the total P content of the DUR3-OE was 8.8% higher and total N content was 4.3% higher than in WT (p < 0.05). Except in pure-urea medium, the engineered strains exhibited significantly increased total P accumulation and superior P recovery efficiency from the culture medium. Transcriptomic analysis revealed that DUR3 overexpression reprograms a coordinated regulatory network associated with N/P metabolism, photosynthesis, and carbon transport pathways. RT-qPCR validation confirmed significant upregulation of PMA2 (plasma membrane H⁺-ATPase), phosphate transporters (PTB3, PTB7), the inorganic carbon transporter HLA3, and photosynthesis-related genes, which was associated with improved nutrient assimilation and photosynthetic performance. These findings establish DUR3 as a key genetic target for engineering microalgae with optimized N-P co-uptake capacity, providing a robust molecular framework for developing high-efficiency algal strains for wastewater bioremediation and nutrient circular economy applications.