Characterization of UDP-Glycosyltransferase Bioparts and Metabolic Engineering of Saccharomyces Cerevisiae to Produce Bioactive Rare Ginsenosides

Ginsenosides, a group of triterpenoids, are the main pharmacological active constituents of ginseng. Most ginsenosides are glycosylated products of the triterpenoid aglycones protopanaxadiol (PPD) and protopanaxatriol (PPT). Rare ginsenosides, such as PPD-derived Compound K (CK), Rh2, Rg3 and PPT-derived F1 and Rh1, typically bear less glycosyl moiety but display a broad range of pharmacological properties, including anti-cancer, anti-inflammation, hepatoprotection and anti-diabetes bioactivities. Despite the available heterologous biosynthesis of their aglycones in yeast, de novo production of ginsenoside by a synthetic biology approach was hindered by the absence of UDP-glycosyltransferase (UGT) bioparts to glucosylate the C3, C6 or C20 hydroxyl of aglycones. We assembled 158 UGT contigs from Panax EST datasets, which were clustered into 59 operational taxonomic units (OTUs). 45 of them (UGTPg1-45) were successfully cloned from Panax ginseng and expressed in Escherichia coli. Through functional screening, UGTs responsible for the production of ginsenosides CK, Rh2, Rg3, F1 and Rh1 were identified and characterized. Based on the UGT bioparts and yeast chassis to produce PPD or PPT, yeast cell factories were built to produce these bioactive rare ginsenosides from glucose. Metabolic engineering and fed-batch fermentation of these strains were performed to further improve the titer. Our study provides a sound manufacturing approach for active rare ginsenosides in yeast via synthetic biology strategies, and reveals the possible biosynthetic pathways of ginsenosides in Panax plants.