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

Abstract

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.

Modular Pathway Compartmentalization for Agroclavine Overproduction in Saccharomyces cerevisiae

“…44 Overexpression of POS5 in S. cerevisiae can increase intracellular NADPH supply overall. For example, carotenoids, 45 squalene, 46 and 7-dehydrocholesterol 47 were overproduced in yeast by overexpressing POS5. Therefore, POS5 was overexpressed in strain Sc158 with an effect on agroclavine production; overexpression of POS5 resulted in an approximately 27.4% increase in agroclavine yield (Figure 4B), which supported the fact that the introduction of the agroclavine biosynthetic pathway into host cells would result in an insufficient supply of NADPH cofactors.…”

Regulation of Ethanol Assimilation for Efficient Accumulation of Squalene in Saccharomyces cerevisiae

“…Competitive pathway inhibition for target product synthesis is commonly used in microbial metabolic engineering studies. 10,13 In order to reduce the competitive downstream consumption of squalene, P ERG1 was replaced by P HXT1 , but the strain suffered from slower growth and a lower squalene titer (Figure 4A). This may be because replacing P ERG1 with P HXT1 decreases the synthesis of downstream steroids in S. cerevisiae since the cell membrane contains various steroids needed to maintain normal physiological activities.…”

“…Therefore, sustainable microbial production of squalene has emerged as an attractive alternative to conventional production methods. In addition, squalene is the precursor of many high-value plant triterpenes and animal steroids. , The currently popular high-value terpenoids and steroids ginsenosides, β-amyrin, diosgenin, and 7-dehydrocholesterol , are synthesized using squalene as a precursor. Stable and sufficient supply of squalene is needed for terpenoid and steroid research and industrial production.…”

Secretory production of 7‐dehydrocholesterol by engineered Saccharomyces cerevisiae

“…[4] Key enzymes that involved in pathways of both mevalonate and post-squalene were characterized and overexpressed by multi-copy integration. [5,6] To balance cell growth and product synthesis, sterol metabolic flux was further remodeled by CRISPRi technology. [7] However, sterols tend to be accumulated intracellularly due to their hydrophobic nature, which could not only induce product inhibitory effects, but also may exert stress on the yeast cells.…”

“…[ 4 ] Key enzymes that involved in pathways of both mevalonate and post‐squalene were characterized and overexpressed by multi‐copy integration. [ 5,6 ] To balance cell growth and product synthesis, sterol metabolic flux was further remodeled by CRISPRi technology. [ 7 ]…”