BACKGROUND : Ginsenoside compound K (CK), one of the primary active metabolites of protopanaxadiol-type ginsenosides, is produced by the intestinal flora that degrade ginseng saponins and exhibits diverse biological properties such as anticancer, anti-inflammatory, and anti-allergic properties. However, it is less abundant in plants. Therefore, enabling its commercialization by construction of a Saccharomyces cerevisiae cell factory is of considerable significance. RESULTS : We induced overexpression of PGM2, UGP1, and UGT1 genes in WLT-MVA5, and obtained a strain that produces ginsenoside CK. The production of CK at 96 h was 263.94 ± 2.36 mg/L, and the conversion rate from protopanaxadiol (PPD) to ginsenoside CK was 64.23 ± 0.41%. Additionally, it was observed that the addition of glycerol was beneficial to the synthesis of CK. When 20% glucose (C mol) in the YPD medium was replaced by the same C mol glycerol, CK production increased to 384.52 ± 15.23 mg/L, which was 45.68% higher than that in YPD medium, and the PPD conversion rate increased to 77.37 ± 3.37% as well. As we previously observed that ethanol is beneficial to the production of PPD, ethanol and glycerol were fed simultaneously in the 5-L bioreactor fed fermentation, and the CK levels reached 1.70 ± 0.16 g/L. CONCLUSIONS : In this study, we constructed an S. cerevisiae cell factory that efficiently produced ginsenoside CK. Glycerol effectively increased the glycosylation efficiency of PPD to ginsenoside CK, guiding higher carbon flow to the synthesis of ginsenosides and effectively improving CK production. CK production attained in a 5-L bioreactor was 1.7 g/L after simultaneous feeding of glycerol and ethanol.