Fungi produce a wide repertoire of secondary metabolites with pharmaceutical impacts on human health. However, the low expression of secondary metabolites limits their clinical application. Triterpenoids produced by Sanghuangporus baumii possess anti-cancer properties, however, their biosynthetic pathway and regulatory mechanism remain unclear. In this study, squalene synthase gene (SbSQS), a key gene involved in the mevalonate pathway, was cloned and analyzed. Sequence analysis identified

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... as a typical squalene synthase with three conserved catalytic structures related to binding and catalysis. The SbSQS gene encodes a 489 amino acid polypeptide with a predicted protein molecular weight of 55.05 kDa. The promoter of SbSQS contains cis-acting regulatory elements involved in the light, abscisic acid and MeJA responsiveness, which is consistent with the promoters of other genes in the mevalonate pathway. Furthermore, SbSQS was cloned into the pET-32a vector and overexpressed in Escherichia coli. During mycelium fermentation of S. baumii, the biomass increased as fermentation progressed, and peaked on day 18 (1.20 g/250 mL). The triterpenoid content did not accumulate during the fermentation process and was 20.8 mg · g−1 on day 18. The SbSQS expression and the triterpenoid content were peaked on day 6, suggesting a correlation between SbSQS expression level with triterpenoid content. This study proposes a key candidate gene for genetic manipulation of the triterpenoid synthesis to enhance the triterpenoid content in S. baumii.