AbstractThe synthesis of vitamin D3 precursor 7‐dehydrocholesterol (7‐DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7‐DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme‐dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7‐DHC from glucose. Then, we predicted five gene knockout targets for 7‐DHC overproduction by the reconstruction of genome‐scale metabolic model. GDH1 gene knockout increased the 7‐DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7‐DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)‐sterol C‐methyltransferase) promoter was screened out. The OD600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7‐DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7‐DHC titer reached 1328 mg/L in 3‐L bioreactor and the specific titer of 7‐DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7‐DHC by systems metabolic engineering.