Spatial Engineering of Yarrowia lipolytica for the Biosynthesis of Capsanthin

Capsanthin is a high-value tetraterpenoid widely utilized in the food, cosmetic, and pharmaceutical industries. However, its traditional production via plant extraction and chemical synthesis faces severe sustainability and cost constraints. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) offers a superior chassis due to its robust mevalonate pathway and abundant lipid droplets for pigment sequestration. In this study, we systematically engineered Y. lipolytica for the de novo biosynthesis of capsanthin. By heterologously co-expressing of Capsicum annuum capsanthin/capsorubin synthase (CaCCS) and Arabidopsis thaliana zeaxanthin epoxidase (AtZEP), we achieved the first reported biosynthesis of capsanthin in Y. lipolytica. Combinatorial screening revealed that full-length sequences of CaCCS and AtZEP yielded optimal production of 6.92 mg/L. To further enhance the productivity of capsanthin, we evaluated spatial compartmentalization strategies. The results demonstrated that targeting the biosynthetic enzymes to the peroxisome via Ser-Lys-Leu (SKL) tags successfully increased the titer to 8.8 mg/L by leveraging its specialized lipophilic and redox microenvironment. Finally, a 5-L fed-batch fermentation was conducted, achieving a maximum capsanthin titer of 48.15 mg/L. This work provides a sustainable and scalable alternative to plant extraction and chemical synthesis for capsanthin production.