High-Yield Production of Novel C₃₀ Carotenoids with an Artificial Biosynthesis Pathway in <i>Yarrowia lipolytica</i>

Traditional microbial research has focused on C₄₀ carotenoids. Recent reports showed that novel C₃₀ carotenoid compounds, found in certain bacterial microorganisms, exhibited antioxidant properties similar to or greater than those of their C₄₀ counterparts. However, the biosynthesis yields were relatively low. In this study, an artificial 8-step pathway for <i>de novo</i> mevalonate biosynthesis in <i>Yarrowia lipolytica</i> was constructed, increasing squalene titer to 1235.79 mg/L. This led to the development of a high-production squalene platform strain capable of supplying sufficient precursors for C₃₀ carotenoid biosynthesis. We identified the low substrate conversion rate as the main limitation through a systematic research. The bottleneck was addressed through protein and compartmentalization engineering, resulting in 90.948 mg/L C₃₀ carotenoid production (11.091 mg/g DCW). Ultimately, by incorporating these approaches, we developed strains capable of producing 2686.77 mg/L C₃₀ carotenoids with a cellular content of 35.05 mg/g DCW in bioreactor fermentations, a significant improvement over the initial strain and the highest ever reported for C₃₀ carotenoids.