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Background <p>The unnatural ginsenoside 3<i>β</i>-<i>O</i>-Glc-DM exhibits potent anti-colon cancer activity. An engineered yeast strain expressing the dammarenediol-II synthase (DS) and glycosyltransferase (PgUGT74AE2) genes from <i>Panax ginseng </i>was previously constructed to produce 3<i>β</i>-<i>O</i>-Glc-DM. However, the titer of 3<i>β</i>-<i>O</i>-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 3<i>β</i>-<i>O</i>-Glc-DM production.</p> Results <p>Molecular 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 3<i>β</i>-<i>O</i>-Glc-DM produced by the strain to 425&#xa0;mg/L. We conducted fed-batch fermentation using a feedback-controlled feeding method in a 3-L bioreactor. The titer of 3<i>β</i>-<i>O</i>-Glc-DM reached 3.4&#xa0;g/L, representing a 42% increase over that achieved by the previously constructed engineered strain Y8CSH (2.4&#xa0;g/L).</p> Conclusions <p>This study significantly enhanced the production of 3<i>β</i>-<i>O</i>-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.</p> Graphical Abstract <p></p>

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Engineering a yeast cell factory for high-level production of unnatural ginsenoside 3β-O-Glc-DM

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月23日

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  • Xiao-Yan Sun,
  • Yu Peng,
  • Ting Gong,
  • Jing-Jing Chen,
  • Tian-Jiao Chen,
  • Jin-Ling Yang,
  • Ping Zhu

摘要

Background

The 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.

Results

Molecular 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).

Conclusions

This 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.

Graphical Abstract