Pathway Refactoring for Efficient 7-Dehydrocholesterol Production in <i>Saccharomyces cerevisiae</i>

7-Dehydrocholesterol (7-DHC) is a subcutaneous sterol and a precursor to various active vitamin D₃. Here, a <i>Saccharomyces cerevisiae</i> strain equipped with the <i>de novo</i> biosynthetic pathway for 7-DHC was constructed. 109.0 mg L^-1 of 7-DHC was achieved initially by introducing heterologous 24-dehydrocholesterol reductase (<i>DHCR24</i>) and overexpressing vital enzymes. Following these modifications, the dynamic regulation of the ergosterol pathway and multicopy expression of <i>DHCR24</i> resulted in an 86.3% increase in the 7-DHC titer. Subsequently, the effects of several organic solvents and surfactants on 7-DHC production were also explored. The addition of ε-polylysine increased the titer of 7-DHC by 99.1%. Finally, by assembling the pathway in peroxisomes and rebalancing the redox levels, the 7-DHC titer reached 517.4 mg L^-1 in shake flasks. Scale-up fermentation with a 5 L bioreactor demonstrated that 3.26 g L^-1 of 7-DHC was produced. The pathway refactoring strategy provides efficient production of 7-DHC in a sustainable manner.