Squalene is a triterpene that can be obtained from fish and plant oils. It is important in cosmetics and vaccines and is a precursor for many high-value terpenes and steroids. In order to increase squalene accumulation, the mevalonate pathway was systematically enhanced. Accumulation of squalene tended to increase when ethanol was added as a carbon source during fermentation, but a high concentration of ethanol affected both the strain growth and accumulation of products. By overexpressing the key trehalose synthesis gene TPS1 and the heat shock protein gene HSP104, the content of trehalose by Saccharomyces cerevisiae (S. cerevisiae) was enhanced, and stress caused by ethanol was relieved. The OD 600 value of the modified S. cerevisiae strain was increased by 80.2%, its ethanol tolerance was increased to 30 g/L, and it retained excellent activity with 50 g/L ethanol. After optimizing the fermentation conditions, the squalene titer in a 5 L bioreactor reached 27.3 g/L and the squalene content was 650 mg/g dry cell weight, the highest squalene production parameters reported to date for a microorganism.

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.