ArticleMicrobial cell factories2026
Engineering a yeast cell factory for high-level production of unnatural ginsenoside 3β-O-Glc-DM.
Article in Microbial cell factories, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundThe unnatural ginsenoside 3β-O-Glc-DM exhibits potent anti-colon cancer activity. An engineered yeast strain expressing the dammarenediol-II synthase (DS) and glycosyltransferase (PgUGT74AE2) genes from Panax ginseng was previously constructed to produce 3β-O-Glc-DM. However, the titer of 3β-O-Glc-DM was insufficient for industrial-scale production. To overcome this limitation, we employed a semi-rational design approach to engineer PgUGT74AE2, integrating enzyme optimization with fermentation enhancement to boost 3β-O-Glc-DM production.
resultsMolecular docking identified 12 key residues near the active pocket as mutation hotspots. Alanine scanning at these positions revealed that substitutions at K276 and H185 improved catalytic activity. Subsequent semi-saturation mutagenesis specifically at K276 yielded mutants K276A and K276V, both exhibiting a 3.4-fold increase in catalytic activity relative to wild-type PgUGT74AE2. This improved activity resulted from reduced steric hindrance and enhanced hydrophobic interactions. The chassis strain Y-ΔHXK2 was optimized for enhanced 2,3-oxidosqualene production by overexpressing key upstream biosynthetic enzymes, down-regulating competitive branch pathways, and overexpressing the transcriptional activator HAC1, thereby generating the strain Y13. Subsequently, the mutant PgUGT74AE2-K276A and DS genes were integrated into Y13 via the CRISPR/Cas9 system to generate the strain Y13-A9. Further optimization of the shake-flask culture conditions increased the titer of 3β-O-Glc-DM produced by the strain to 425 mg/L. We conducted fed-batch fermentation using a feedback-controlled feeding method in a 3-L bioreactor. The titer of 3β-O-Glc-DM reached 3.4 g/L, representing a 42% increase over that achieved by the previously constructed engineered strain Y8CSH (2.4 g/L).
conclusionsThis study significantly enhanced the production of 3β-O-Glc-DM in the engineered yeast through protein and metabolic engineering. It lays a foundation for its industrial-scale production and the development of new anti-colon cancer drugs.
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