AUTHOR=Wei Wenqian , Gao Song , Yi Qiong , Liu Anjian , Yu Shiqin , Zhou Jingwen 

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

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.978074

DOI=10.3389/fmicb.2022.978074

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Saccharomyces cerevisiae</italic>. Firstly, the biosynthesis pathway was constructed by knocking out the competitive pathway genes <italic>ERG5</italic> and <italic>ERG6</italic> and integrating two <italic>DHCR24</italic> copies from <italic>Gallus gallus</italic> at both sites. Then, 7-DHC titer was improved by knocking out <italic>MOT3</italic>, which encoded a transcriptional repressor for the 7-DHC biosynthesis pathway. Next, by knocking out <italic>NEM1</italic> and <italic>PAH1</italic>, 7-DHC accumulation was improved, and genes upregulation was verified by quantitative PCR (qPCR). Additionally, <italic>tHMG1</italic>, <italic>IDI1</italic>, <italic>ERG2</italic>, <italic>ERG3</italic>, <italic>DHCR24</italic>, <italic>POS5</italic>, and <italic>CTT1</italic> 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 <italic>de novo</italic> 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.</p>