Modular remodeling of sterol metabolism for overproduction of 7-dehydrocholesterol in engineered yeast

Vitamin D₃ is a fat-soluble vitamin essential for the human body, and the biosynthesis of its precursor, 7-dehydrocholesterol (7-DHC), gains extensive attention. In this work, six genes (tHMG1, IDI1, ERG1, ERG11, ADH2, ERG7) and a transcription factor mutant UPC2^G888A were overexpressed, increasing the 7-DHC titer from 1.2 to 115.3 mg/L. The CRISPR-mediated activation and repression systems were constructed and applied to the synthesis of 7-DHC, increasing the 7-DHC titer to 312.4 mg/L. Next, enzymes were compartmentalized into the endoplasmic reticulum (ER) and the ER lumen was enlarged by overexpressing INO2. The 7-DHC titer of the finally engineered yeast reached 455.6 mg/L in a shake flask and 2870 mg/L in a 5 L bioreactor, the highest 7-DHC titer reported so far. Overall, this study achieved a highly efficient 7-DHC synthesis by remodeling the complicated sterol synthesis modules, paving the way for large-scale 7-DHC bioproduction in the future.