Characterization of Panax ginseng UDP-Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rh1 in Metabolically Engineered Yeasts

Abstract

Ginsenosides, the main pharmacologically active natural compounds in ginseng (Panax ginseng), are mostly the glycosylated products of protopanaxadiol (PPD) and protopanaxatriol (PPT). No uridine diphosphate glycosyltransferase (UGT), which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPg1, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside F1. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity (>84%) with UGTPg1. We demonstrate that UGTPg100 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rh1, and UGTPg101 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rg1 from F1. However, UGTPg102 and UGTPg103 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rh1 by introducing the genetically engineered PPT-producing pathway and UGTPg1 or UGTPg100. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

Transcriptome profiling shows gene regulation patterns in ginsenoside pathway in response to methyl jasmonate in Panax Quinquefolium adventitious root

“…In general, glycosylation is the last step in the biosynthesis of secondary metabolites. Recently, identified eight GTs (UGT73C11, UGT73C10, UGT74AE2, UGT94Q2, UGT71A27, UGTPg1, UGTPg100, UGTPg101) involved in the later steps of ginsenoside biosynthesis in the closely related species P. ginseng 101112.…”

“…It is known that ginsenoside biosynthesis is achieved mainly through three reaction steps of 2, 3-oxidosqualene: cyclization, hydroxylation and glucosidation7. However, despite these advances, further elucidation of the ginsenoside biosynthetic pathways has been slowed by the limited sequence information available for cytochrome P450 (CYP450) (CYP716A53v2, CYP716A47, CYP716A52v2)89 and glycosyltransferase (GT) (UGT74AE2, UGT94Q2, UGT71A27, UGTPg1, UGTPg100, UGTPg101)101112.…”

Effect of temperature on morphology, ginsenosides biosynthesis, functional genes, and transcriptional factors expression in Panax ginseng adventitious roots

“…Dammarenediol II next is transformed to protopanaxadiol and protopanaxatriol by two cytochrome P450 (CYP) genes (CYP716A47, CYP716A53v2) (Han, Hwang, Choi, Kim, & Choi, ; Han, Kim, Kwon, & Choi, ). Then, protopanaxatriol and protopanaxatriol, transformed by UDP‐glycosyltransferases (UGT) genes UGT94Q2 and UGTpg100 could produce Rh 1 and Rh 2 (Figure S1; Jung et al, ; Wei et al, ).…”

Integrated Transcriptomic and Metabolomic Analysis of Five Panax ginseng Cultivars Reveals the Dynamics of Ginsenoside Biosynthesis

“…Candidate genes responsible for ginsenoside biosynthesis in the Panax genus have been identified in P. ginseng (Han et al, 2011, 2012; Jung et al, 2014; Kim et al, 2014; Jayakodi et al, 2015; Wang P. et al, 2015; Wei et al, 2015), P. notoginseng (Liu et al, 2015; Rai et al, 2016), and P. vietnamensis (Zhang et al, 2015). Transcriptome analysis of P. notoginseng identified several candidate unigenes encoding cytochrome P450 and glycosyltransferase and revealed that these genes were conserved across all Panax species (Rai et al, 2016).…”