A two-prong mutagenesis and adaptive evolution strategy to enhance the temperature tolerance and productivity ofNannochloropsis oculata.

Incorporation of microalgae in biorefineries intended to help society reach carbon neutrality is hindered by algal growth inhibition at high temperatures, necessitating the use of costly and carbon-intensive cooling systems. In the present study, a two-prong strategy of random mutagenesis and adaptive laboratory evolution to generate robust thermotolerant strains of Nannochloropsis oculata, was used. The best mutants demonstrated increased productivity at 35°C, which was 10°C higher than the optimal temperature of the wild type. In a 2-L photobioreactor at 35°C, biomass and lipid productivity were 1.43-fold and 2.24-fold higher, respectively, than wild type at 25°C. Higher pigment and carbohydrate content contributed to the mutants' rapid growth and enhanced photosynthetic efficiency. Metabolomics and lipidomics showed rewiring of the central carbon metabolism and membrane lipid synthesis in thermotolerant strains to ensure cellular homeostasis without compromising productivity. Tagatose and phosphatidylethanolamine upregulation were identified as future genetic targets for further enhancing lipid production.