Suppressing Glycerol‐3‐phosphate Phosphatase and Enhancing Glycerol‐3‐Phosphate Shuttle Flux Crucial for High‐Efficiency Fatty Acid Production in the Fast‐Growing Oleaginous <i>Schizochytrium</i>

The industrial microbial production of fatty acids is limited by low titers and productivities, highlighting the need for chassis engineering and mechanistic insights into efficient metabolic pathways. The heterotrophic microalga Schizochytrium offers distinct advantages, and Schizochytrium ZW1 is engineered into a high-efficiency fatty acid production chassis. A fluorescence-activated cell sorting strategy is developed to overcome cellular aggregation, enabling the rapid isolation of Schizochytrium ZW2, which exhibited enhanced growth and fatty acid accumulation. Multiomics analysis of ZW2 revealed that mutations suppressing glycerol-3-phosphate phosphatase activity promoted triacylglycerol synthesis, and mutations in the polyunsaturated fatty acid synthase C subunit increased polyunsaturated fatty acid production. Furthermore, increased glycerol-3-phosphate shuttle flux in ZW2 enhanced both biomass and fatty acid accumulation. The crucial roles of these mutations are confirmed by introducing modifications to enhance glycerol-3-phosphate shuttle and suppress glycerol-3-phosphate phosphatase activity in ZW1, which yielded ≈80% of the fatty acid production of ZW2. Further engineering generated Schizochytrium ZW2-D-L2, which achieved total fatty acid and triacylglycerol titers of 94.65 and 88.51 g L^-1, and productivities of 1.39 and 1.30 g L^-1 h^-1, respectively. This performance doubles that of ZW1 and surpasses microbial benchmarks. This work elucidates regulatory mechanisms and establishes Schizochytrium as a scalable platform for industrial biotechnology.