Key Glycosyltransferase Genes of Panax notoginseng: Identification and Yeast Construction of Rare Ginsenosides

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg(3) and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg(3) reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Automated summary

In this study, candidate glycosyltransferase genes were screened based on the previous genome and transcriptome data of Panax notoginseng, and the full length of 27 UGT genes were successfully cloned. Among them, it was found that the PnUGT33 gene could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides. The enzymatic kinetics of PnUGT33 were analyzed and the catalytic mechanism was predicted through molecular docking simulation. Ultimately, the biosynthetic pathway of rare ginsenoside Rg(3) and gypenoside LXXV was reconstructed in yeast, and the yields were significantly improved by overexpressing UDPG biosynthetic genes.