Sequential UV mutagenesis and CO2 acclimation reprogram metabolic carbon allocation in Euglena gracilis.

Euglena gracilis is promising for CO2 bioutilization, but its metabolic response to elevated CO2 is poorly characterized. Here, the LE-ZW mutant, obtained via UV mutagenesis and CO2 acclimation, was evaluated under 5% and 10% CO2. LE-ZW exhibited 7% higher cell density and a 61% increase in photosynthetic carbon fixation at 10% CO2 compared with 5% CO2. Carbon allocation shifted with CO2 concentration: lipid accumulation peaked at 20.57% under 5% CO2, while paramylon content reached 37.37% under 10% CO2. Protein levels increased by 4% under high CO2 (vs 5% CO2 in LE-ZW). Transcriptomic and metabolomic analyses revealed extensive metabolic reprogramming: vs the wild type at 5% CO2, LE-ZW had 923 upregulated genes and 243 increased metabolites (enriched in TCA cycle and fatty acid biosynthesis, including 1.38-fold higher eicosapentaenoic acid); vs 5% CO2 at 10%, it had 396 upregulated genes and 20 increased metabolites (enriched in Calvin cycle, GS-GOGAT pathway, and aminoacyl-tRNA biosynthesis). These changes identified regulatory hubs (glycolysis-gluconeogenesis nodes, acetyl-CoA supply, glucose-6-phosphate partitioning) that redirected carbon flux between lipids and paramylon. Antioxidant defense was enhanced (29% higher superoxide dismutase activity). These results demonstrate LE-ZW's integrated regulation of carbon-nitrogen metabolism and redox homeostasis under high CO2, supporting its potential for industrial CO2 mitigation and bio-based production.