Reengineering of 7-dehydrocholesterol biosynthesis in 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 . Firstly, the biosynthesis pathway was constructed by knocking out the competitive pathway genes and and integrating two copies from at both sites. Then, 7-DHC titer was improved by knocking out , which encoded a transcriptional repressor for the 7-DHC biosynthesis pathway. Next, by knocking out and , 7-DHC accumulation was improved, and genes upregulation was verified by quantitative PCR (qPCR). Additionally, , , , , , , and 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 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.