Multiple Strategies Enhance 7-Dehydrocholesterol Production from Kitchen Waste by Engineered <i>Yarrowia lipolytica</i>

7-Dehydrocholesterol (7-DHC) is an important precursor of vitamin D₃. The microbial synthesis of 7-DHC has attracted substantial attention. In this study, multiple strategies were developed to create a sustainable green route for enhancing 7-DHC yield from kitchen waste by engineered <i>Yarrowia lipolytica</i>. <i>Y. lipolytica</i> strains were engineered and combined with various Δ²⁴-dehydrocholesterol reductases. Overexpressing all the genes in the mevalonate pathway improved the precursor pool, increasing the 7-DHC titer from 21.8 to 145.6 mg/L. Additionally, optimizing medium components using the response surface method significantly raised the 7-DHC titer to 391.0 mg/L after shake flask cultivation. The engineered strain yielded a record 7-DHC titer of 3.5 g/L in a 5-L bioreactor when kitchen waste was used as a carbon source. Overall, these results demonstrate that engineered <i>Y. lipolytica</i> efficiently synthesizes 7-DHC from waste lipid feedstock, offering a promising route for its bioproduction.