Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3−OH and/or C20− OH of protopanaxadiol (PPD) and C6−OH and/or C20−OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→ 2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc 2 -DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc 2 -DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc 2 -DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc 2 -DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc 2 -DM as a potential cardioprotective drug candidate.