Enhancing Cordycepin Biosynthesis in <i>Yarrowia lipolytica</i> via Lipid Droplets Compartmentalization Engineering and Optimized Fermentation Strategies

Cordycepin, a physiologically active nucleoside compound with broad applications in healthcare, is biosynthesized in <i>Cordyceps militaris</i> through a protein complex formed by CmCns1 and CmCns2. To enhance cordycepin heterologous production in <i>Yarrowia lipolytica</i>, this study confirmed the colocalization of CmCns1 and CmCns2 on lipid droplets, with CmCns1 dominating this process by recruiting CmCns2 from the cytoplasm to lipid droplets via strong interactions. Critical lipid-droplet-targeting motifs within CmCns1 were identified. On this basis, an engineered strain YL-CD3 was developed by expanding the lipid droplets and CmCns3-NK compartmentalization. Then, the fermentation parameters were optimized to increase the yield of cordycepin to 2008.23 mg/L in shake flasks. Finally, fed-batch fermentation in a 2.4 L bioreactor for 144 h achieved 4780.75 mg/L (150.1 mg/OD₆₀₀ and 66.57 mg/g glucose), marking the highest reported titer in <i>Y. lipolytica</i>. This work establishes <i>Y. lipolytica</i> as a high-potential platform for efficient cordycepin biosynthesis.