Reengineering of 7-dehydrocholesterol biosynthesis in Saccharomyces cerevisiae using combined pathway and organelle strategies

7-Dehydrocholesterol (7-DHC) is a widely used sterol and a precursor of several costly steroidal drugs. In this study, 7-DHC biosynthesis pathway was constructed and modified in Saccharomyces cerevisiae . Firstly, the biosynthesis pathway was constructed by knocking out the competitive pathway genes ERG5 and ERG6 and integrating two DHCR24 copies from Gallus gallus at both sites. Then, 7-DHC titer was improved by knocking out MOT3 , which encoded a transcriptional repressor for the 7-DHC biosynthesis pathway. Next, by knocking out NEM1 and PAH1 , 7-DHC accumulation was improved, and genes upregulation was verified by quantitative PCR (qPCR). Additionally, tHMG1 , IDI1 , ERG2 , ERG3 , DHCR24 , POS5 , and CTT1 integration into multi-copy sites was used to convert precursors to 7-DHC, and increase metabolic flux. Finally, qPCR confirmed the significant up-regulation of key genes transcriptional levels. In a 96 h shaker flask fermentation, the 7-DHC titer was 649.5 mg/L by de novo synthesis. In a 5 L bioreactor, the 7-DHC titer was 2.0 g/L, which was the highest 7-DHC titer reported to date. Our study is of great significance for the industrial production of 7-DHC and steroid development for medical settings.