Engineering hypo-osmotic Crypthecodinium cohnii via SL-ALE for high-yield DHA biosynthesis from agricultural byproducts

The marine dinoflagellate Crypthecodinium cohnii has emerged as a promising industrial producer of omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). However, its high salt requirements pose significant challenges for industrial-scale cultivation, with the underlying salinity adaptation mechanisms remaining poorly characterized. This study developed an innovative solid-liquid biphasic cultivation system for low-salinity adaptive laboratory evolution, utilizing broken rice hydrolysate (BRH) as a cost-effective carbon source. Through systematic selection pressure, we obtained the evolved strain LS8 exhibiting enhanced low-salinity tolerance. When cultivated under optimized conditions (8 g/L sea salt), LS8 demonstrated remarkable PUFAs biosynthetic capacity, achieving a 1.018 g/L DHA yield, representing a 1.13-fold increase compared to conventional methods. Mechanistic analysis revealed that improved NADPH availability through enhanced malic enzyme activity and upregulation of polyketide synthase pathway genes synergistically contributed to superior lipid accumulation. Nutrient optimization studies identified that a medium formulation containing 45 % BRH supplemented with 2 g/L yeast extract maximized DHA production while reducing sea salt consumption by 68 %. These findings establish LS8 as an industrially superior candidate for sustainable DHA production, offering significant advantages in operational cost reduction and environmental compatibility through substantially decreased salt requirements. Our integrated approach combining adaptive evolution with biorefinery-based nutrient utilization provides a strategic framework for optimizing marine microorganism cultivation in low-salinity bioprocessing systems.