Efficient 7‑Dehydrocholesterol\nProduction by\nMultiple Metabolic Engineering of Diploid Saccharomyces\ncerevisiae

7-Dehydrocholesterol (7-DHC), a direct precursor of vitamin\nD3,\nhas attracted increasing attention in microbial fermentation recently.\nIn this study, 7-DHC biosynthesis in diploid Saccharomyces\ncerevisiae with robust ergosterol production was achieved\nby heterologous 24-dehydrocholesterol reductase expression, generating\n44.1 mg/L 7-DHC, whereas the titer of ergosterol decreased by 40.5%.\nThe ergosterol biosynthetic pathway was completely blocked by knocking\nout <i>ERG6</i> and <i>ERG5</i>, affording a 4.2-fold\nincrease in the 7-DHC titer. Subsequently, the facilitation of the\nmevalonate and the postsqualene pathways accompanied by elimination\nof transcriptional repressors enhanced 7-DHC synthesis, and the 7-DHC\ntiter reached 738.5 mg/L in a shake flask. Further validation in a\n50 L fermenter demonstrated that the 7-DHC titer reached 3.80 g/L\nwithin just 24 h, with productivity reaching 158.3 mg/L/h, setting\na new benchmark as the highest reported to date. This study paves\nthe way toward a large-scale and cost-effective manufacture of 7-DHC.