title: 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.
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Wang
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Qunfang
authors.initials: Q
authors.lastname: Liu
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Chengshuai
authors.initials: C
authors.lastname: Yang
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China. Electronic address: jmyue@mail.shcnc.ac.cn.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Yue
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address: yanxing@sibs.ac.cn.
authors.forename: Xing
authors.initials: X
authors.lastname: Yan
authors.affiliation: CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address: zhouzhihua@sippe.ac.cn.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D017343:Genes, Plant; D005818:Genetic Engineering; D036145:Ginsenosides / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D060847:Metabolic Engineering; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D005894:Panax / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012502:Sapogenins / Q000378:metabolism*; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D000596:Amino Acids; D036145:Ginsenosides; D050505:Mutant Proteins; D012502:Sapogenins; C081552:protopanaxatriol; C055327:ginsenoside F1; D014530:Uridine Diphosphate; C425564:ginsenoside Rh1; D016695:Glycosyltransferases
keywords: Panax ginseng; UDP-glycosyltransferase; ginsenoside F1; ginsenoside Rh1; protopanaxatriol; triterpenoids