Construction and\nOptimization of a Yeast Cell Factory\nfor Producing Active Unnatural Ginsenoside 3β‑<i>O</i>‑Glc<sup>2</sup>‑DM

Ginsenosides are major active components of Panax\nginseng, which are generally glycosylated at C3–OH\nand/or C20–OH of protopanaxadiol (PPD) and C6–OH and/or\nC20–OH of protopanaxatriol. However, the glucosides of dammarenediol-II\n(DM), which is the direct precursor of PPD, have scarcely been separated\nfrom P. ginseng. Because different\npositions and numbers of the hydroxyl and glycosyl groups lead to\na diversity of structure and function of the ginsenosides, it can\nbe inferred that DM glucosides may have different pharmacological\nactivities compared with natural ginsenosides. Herein, we first constructed\nthe cell factory for <i>de novo</i> biosynthesis of 3-<i>O</i>-(β-<i>D</i>-glucopyranosyl-(1→2)-β-<i>D</i>-glucopyranosyl)-dammar-24-ene-3β,20<i>S</i>-diol (3β-<i>O</i>-Glc<sup>2</sup>-DM) by introducing\nthe codon-optimized genes encoding dammarenediol-II synthase, two\nUDP-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-<i>O</i>-Glc<sup>2</sup>-DM was then increased from 18.9 to 148.0 mg/L by\nseveral metabolic engineering strategies including overexpressing\nthe rate-limiting enzymes of triterpenoid biosynthesis, balancing\ncarbon flux of biosynthetic pathways of triterpenoid and ergosterol,\nand engineering endoplasmic reticulum. Furthermore, the 3β-<i>O</i>-Glc<sup>2</sup>-DM titer of 766.3 mg/L was achieved through\nfed-batch fermentation in a 3-L bioreactor. Finally, <i>in vitro</i> assays demonstrated that 3β-<i>O</i>-Glc<sup>2</sup>-DM exhibited a protective effect on H/R-induced cardiomyocyte damage.\nThis work provides a feasible approach for production of 3β-<i>O</i>-Glc<sup>2</sup>-DM as a potential cardioprotective drug\ncandidate.