Combinatorial metabolic engineering of Saccharomyces cerevisiae for improved production of 7-dehydrocholesterol

7-Dehydrocholesterol (7-DHC), a key pharmaceutical intermediate in the production of vitamin D₃, has a wide range of applications. To explore fermentative synthesis of 7-DHC, a 7-DHC-producing Saccharomyces cerevisiae strain was constructed by blocking the competitive pathway, eliminating rate-limiting steps, altering global regulation, and pathway compartmentalization. After blocking the competitive pathway by disrupting ERG5 and ERG6 and introducing DHCR24 from Gallus gallus, S. cerevisiae produced 139.72 mg/L (17.04 mg/g dry cell weight, hereafter abbreviated as DCW) 7-DHC. Subsequent alteration of global regulation by deleting ROX1 and overexpressing UPC2-1 increased 7-DHC production to 217.68 mg/L (37.56 mg/g DCW). To remove the accumulated squalene, the post-squalene pathway was strengthened by co-overexpression of P -driven ERG11 and P -driven ERG1, which improved 7-DHC titer and yield to 281.73 mg/L and 46.78 mg/g DCW, respectively, and reduced squalene content by 90.12%. We surmised that the sterol precursors in the plasma membrane and peroxisomes may not be accessible to the pathway enzymes, thus we re-localized DHCR24p and Erg2p-GGGGS-Erg3p to the plasma membrane and peroxisomes, boosting 7-DHC production to 357.53 mg/L (63.12 mg/g DCW). Iron supplementation further increased 7-DHC production to 370.68 mg/L in shake flasks and 1.56 g/L in fed-batch fermentation. This study demonstrates the power of global regulation and subcellular relocalization of key enzymes to improve 7-DHC synthesis in yeast.