Harnessing a Nonconventional Yeast for 7-Dehydrocholesterol Overproduction

Vitamin D3 is critical for human health. Its precursor, 7-dehydrocholesterol (7-DHC), can be microbially synthesized as a sustainable alternative to traditional chemical synthesis, with advantages that include environmental friendliness, reduced pollution, and the use of low-cost raw materials. This study investigated the potential of an unconventional yeast, Candida tropicalis, as a novel platform for 7-DHC production. Applying codon optimization and by integrating the DHCR24 gene into C. tropicalis, a chassis strain for 7-DHC biosynthesis was successfully constructed. Enhancing the squalene supply and alleviating postsqualene pathway bottlenecks increased the 7-DHC titer from 10.1 to 89.5 mg/L. Suppressing the ergosterol branch pathway by knocking out ERG5 and downregulating ERG6 combined with increasing the DHCR24 gene copy number increased the titer to 365.7 mg/L. Additionally, relocating ERG7 to the endoplasmic reticulum (ER) coupled with ER expansion engineering further increased the 7-DHC titer to 432.3 mg/L. By optimizing the fermentation medium and scaling up the process, the C. tropicalis ICE2-INO2 strain ultimately achieved a 7-DHC titer of 3.65 g/L in a 5-L bioreactor. Collectively, this work established C. tropicalis as a novel and efficient yeast platform for 7-DHC biosynthesis and demonstrated the effectiveness of applying metabolic engineering methods.