Abstract
Main conclusion
Two UDP-glycosyltransferases from Panax vienamensis var. fuscidiscus involved in ocotillol-type ginsenoside MR2 (majonside-R2) biosynthesis were identified. PvfUGT1 and PvfUGT2 sequentially catalyzes 20S,24S-Protopanxatriol Oxide II and 20S,24R-Protopanxatriol Oxide I to pseudoginsenoside RT4/RT5 and RT4/RT5 to 20S, 24S-MR2/20S, 24S-MR2.
Abstract
Ocotilol type saponin MR2 (majonside-R2) is the main active component of Panax vietnamensis var. fuscidiscus (commonly known as ‘jinping ginseng’) and is well known for its diverse pharmacological activities. The use of MR2 in the pharmaceutical industry currently depends on its extraction from Panax species. Metabolic engineering provides an opportunity to produce high-value MR2 by expressing it in heterologous hosts. However, the metabolic pathways of MR2 remain enigmatic, and the two-step glycosylation involved in MR2 biosynthesis has not been reported. In this study, we used quantitative real-time PCR to investigate the regulation of the entire ginsenoside pathway by MeJA (methyl jasmonate), which facilitated our pathway elucidation. We found six candidate glycosyltransferases by comparing transcriptome analysis and network co-expression analysis. In addition, we identified two UGTs (PvfUGT1 and PvfUGT2) through in vitro enzymatic reactions involved in the biosynthesis of MR2 which were not reported in previous studies. Our results show that PvfUGT1 can transfer UDP-glucose to the C6-OH of 20S, 24S-protopanaxatriol oxide II and 20S, 24R-protopanaxatriol oxide I to form pseudoginsenoside RT4 and pseudoginsenoside RT5, respectively. PvfUGT2 can transfer UDP-xylose to pseudoginsenoside RT4 and pseudoginsenoside RT5 to form 20S, 24S-MR2 and 20S, 24S-MR2. Our study paves the way for elucidating the biosynthesis of MR2 and producing MR2 by synthetic biological methods.
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Data availability
Supplementary materials for this article can be found on line, and all transcriptome data were deposited at the NCBI Sequence Read Archive (SRA) (accession number: PRJNA938799).
Abbreviations
- CYP:
-
Cytochrome P450 monooxygenase
- DDS:
-
Dammarane-II synthase
- MeJA:
-
Methyl-jasmonate
- MR2:
-
Majonoside-R2
- OCT:
-
Ocotillol
- PPD:
-
Protopanaxadiol
- PPT:
-
Protopanaxatriol
- SE:
-
Squalene epoxidase
- SS:
-
Squalene synthase
- UGT:
-
Uridine diphosphate glycosyltransferase
References
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Bönisch F, Frotscher J, Stanitzek S, Rühl E, Wüst M, Bitz O, Schwab W (2014) A UDP-glucose:monoterpenol glucosyltransferase adds to the chemical diversity of the grapevine metabolome. Plant Physiol 165(2):561–581. https://doi.org/10.1104/pp.113.232470
Bowles D, Isayenkova J, Lim E-K, Poppenberger B (2005) Glycosyltransferases: managers of small molecules. Curr Opin Plant Biol 8(3):254–263. https://doi.org/10.1016/j.pbi.2005.03.007
Christensen LP (2009) Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res 55:1–99. https://doi.org/10.1016/S1043-4526(08)00401-4
Deng Z-L, Münch PC, Mreches R, McHardy AC (2022) Rapid and accurate identification of ribosomal RNA sequences via deep learning. Nucleic Acids Res 50(10):e60. https://doi.org/10.1093/nar/gkac112
Han J-Y, Kim H-J, Kwon Y-S, Choi Y-E (2011) The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 52(12):2062–2073. https://doi.org/10.1093/pcp/pcr150
Han J-Y, Hwang H-S, Choi S-W, Kim H-J, Choi Y-E (2012) Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 53(9):1535–1545. https://doi.org/10.1093/pcp/pcs106
Haralampidis K, Trojanowska M, Osbourn AE (2002) Biosynthesis of triterpenoid saponins in plants. Adv Biochem Eng Biotechnol 75:31–49. https://doi.org/10.1016/j.pbi.2005.03.007
Hayashi H, Huang P, Inoue K (2003) Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol 44(4):404–411. https://doi.org/10.1093/pcp/pcg054
Hendrawati O, Yao Q, Kim HK, Linthorst HJ, Erkelens C, Lefeber AW, Choi YH, Verpoorte R (2006) Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy. Plant Sci 170(6):1118–1124. https://doi.org/10.1016/j.plantsci.2006.01.017
Hou M, Wang R, Zhao S, Wang Z (2021) Ginsenosides in Panax genus and their biosynthesis. Acta Pharm Sin B 11(7):1813–1834. https://doi.org/10.1016/j.apsb.2020.12.017
Hu FX, Zhong JJ (2008) Jasmonic acid mediates gene transcription of ginsenoside biosynthesis in cell cultures of Panax notoginseng treated with chemically synthesized 2-hydroxyethyl jasmonate. Process Biochem 43(1):113–118. https://doi.org/10.1016/j.procbio.2007.10.010
Huong NT, Matsumoto K, Yamasaki K, Duc NM, Nham NT, Watanabe H (1997) Majonoside-R2, a major constituent of Vietnamese ginseng, attenuates opioid-induced antinociception. Pharmacol Biochem Behav 57(1–2):285–291. https://doi.org/10.1016/s0091-3057(96)00348-6
Itkin M, Davidovich-Rikanati R, Cohen S, Portnoy V, Doron-Faigenboim A, Oren E, Freilich S, Tzuri G, Baranes N, Shen S, Petreikov M, Sertchook R, Ben-Dor S, Gottlieb H, Hernandez A, Nelson DR, Paris HS, Tadmor Y, Burger Y, Lewinsohn E, Katzir N, Schaffer A (2016) The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii. Proc Natl Acad Sci USA 113(47):E7619–E7628. https://doi.org/10.1073/pnas.1604828113
Jung S-C, Kim W, Park SC, Jeong J, Park MK, Lim S, Lee Y, Im W-T, Lee JH, Choi G, Kim SC (2014) Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant Cell Physiol 55(12):2177–2188. https://doi.org/10.1093/pcp/pcu147
Kim OT, Bang KH, Kim YC, Dong YH, Min YK, Cha SW (2009) Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate. Plant Cell Tissue Organ Cult 98(1):25–33. https://doi.org/10.1007/s11240-009-9535-9
Kim OTP, Le MD, Trinh HX, Nong HV (2016) In silico studies for the interaction of tumor necrosis factor-alpha (TNF-α) with different saponins from Vietnamese ginseng (Panax vietnamesis). Biophys Physicobiol 13:173–180. https://doi.org/10.2142/biophysico.13.0_173
Konoshima T, Takasaki M, Ichiishi E, Murakami T, Tokuda H, Nishino H, Duc NM, Kasai R, Yamasaki K (1999) Cancer chemopreventive activity of majonoside-R2 from Vietnamese ginseng. Panax Vietnamensis Cancer Lett 147(1–2):11–16. https://doi.org/10.1016/s0304-3835(99)00257-8
Kurze E, Wüst M, Liao J, McGraphery K, Hoffmann T, Song C, Schwab W (2022) Structure-function relationship of terpenoid glycosyltransferases from plants. Natural Product Rep 39(2):389–409. https://doi.org/10.1039/d1np00038a
Le THV, Lee GJ, Vu HKL, Kwon SW, Nguyen NK, Park JH, Nguyen MD (2015) Ginseng saponins in different parts of Panax vietnamensis. Chem Pharmaceut Bull 63(11):950–954. https://doi.org/10.1248/cpb.c15-00369
Lee M-H, Jeong J-H, Seo J-W, Shin C-G, Kim Y-S, In J-G, Yang D-C, Yi J-S, Choi Y-E (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45(8):976–984. https://doi.org/10.1093/pcp/pch126
Li X, Wang Y, Fan Z, Wang Y, Wang P, Yan X, Zhou Z (2021) High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae. Metab Eng 66:87–97. https://doi.org/10.1016/j.ymben.2021.04.006
Li Y, Baldauf S, Lim EK, Bowles DJ (2001) Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. J Biol Chem 276(6):4338–4343. https://doi.org/10.1074/jbc.M007447200
Li Y, Lin Y, Jia B, Chen G, Shi H, Xu R, Li X, Tang J, Tang Q, Zhang G (2022) Transcriptome analysis reveals molecular mechanisms underlying methyl jasmonate-mediated biosynthesis of protopanaxadiol-type saponins in Panax notoginseng leaves. J Plant Biol 65(1):29–41. https://doi.org/10.1007/s12374-021-09331-4
Lim E-K, Higgins GS, Li Y, Bowles DJ (2003) Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta. Biochem J 373(Pt 3):987–992. https://doi.org/10.1042/BJ20021453
Lu C, Zhao SJ, Wang XS (2017) Functional regulation of a UDP-glucosyltransferase gene (Pq3-O-UGT1) by RNA interference and overexpression in Panax quinquefolius. Plant Cell Tissue Organ Cult 129(3):445–456
Ma CH, Jiang NH, Deng MH, Chen JW, Zhang GH (2016) Cloning and characterization of three squalene epoxidase genes in Panax vietnamensis var. fuscidicus, a rare medicinal plant with high content of ocotillol-type ginsenosides. Pakistan J Bot 48(6):2453–2465
Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Bélanger A, Fournel-Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF, Nebert DW (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7(4):255–269. https://doi.org/10.1097/00008571-199708000-00001
Nguyen MD, Nguyen TN, Kasai R, Ito A, Yamasaki K, Tanaka O (1993) Saponins from Vietnamese ginseng, Panax vietnamensis Ha et Grushv. collected in central Vietnam I. Chem Pharm Bull 41(11):2010–2014. https://doi.org/10.1248/cpb.41.2010
Niu Y, Luo H, Sun C, Yang T-J, Dong L, Huang L, Chen S (2014) Expression profiling of the triterpene saponin biosynthesis genes FPS, SS, SE, and DS in the medicinal plant Panax notoginseng. Gene 533(1):295–303. https://doi.org/10.1016/j.gene.2013.09.045
Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14(4):417–419. https://doi.org/10.1038/nmeth.4197
Rajabian A, Rameshrad M, Hosseinzadeh H (2019) Therapeutic potential of Panax ginseng and its constituents, ginsenosides and gintonin, in neurological and neurodegenerative disorders: a patent review. Expert Opin Ther Pat 29(1):55–72. https://doi.org/10.1080/13543776.2019.1556258
Ross J, Li Y, Lim E, Bowles DJ (2001) Higher plant glycosyltransferases. Genome Biol 2(2):REVIEWS3004. https://doi.org/10.1186/gb-2001-2-2-reviews3004
Ru W, Wang D, Xu Y, He X, Sun Y-E, Qian L, Zhou X, Qin Y (2015) Chemical constituents and bioactivities of Panax ginseng (C. A. Mey). Drug Discov Ther 9(1):23–32. https://doi.org/10.5582/ddt.2015.01004
Shan H, Segura MJR, Wilson WK, Lodeiro S, Matsuda SPT (2005) Enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes. J Amer Chem Soc 127(51):18008–18009. https://doi.org/10.1021/ja055822g
Suzuki H, Achnine L, Xu R, Matsuda SPT, Dixon RA (2002) A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula. Plant J 32(6):1033–1048. https://doi.org/10.1046/j.1365-313x.2002.01497.x
Tang Q-Y, Chen G, Song W-L, Fan W, Wei K-H, He S-M, Zhang G-H, Tang J-R, Li Y, Lin Y, Yang S-C (2019) Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides. Planta 249(2):393–406. https://doi.org/10.1007/s00425-018-2995-6
Thu VT, Yen NTH, Tung NH, Bich PT, Han J, Kim HK (2021) Majonoside-R2 extracted from Vietnamese ginseng protects H9C2 cells against hypoxia/reoxygenation injury via modulating mitochondrial function and biogenesis. Bioorg Med Chem Lett 36:127814. https://doi.org/10.1016/j.bmcl.2021.127814
Tran QL, Adnyana IK, Tezuka Y, Nagaoka T, Tran QK, Kadota S (2001) Triterpene saponins from Vietnamese ginseng (Panax vietnamensis) and their hepatocytoprotective activity. J Nat Prod 64(4):456–461. https://doi.org/10.1021/np000393f
Wang D, Wang J, Shi Y, Li R, Fan F, Huang Y, Li W, Chen N, Huang L, Dai Z, Zhang X (2020) Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform. Metab Eng 61:131–140. https://doi.org/10.1016/j.ymben.2020.05.007
Wang P, Wei W, Ye W, Li X, Zhao W, Yang C, Li C, Yan X, Zhou Z (2019) Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency. Cell Discovery 5:5. https://doi.org/10.1038/s41421-018-0075-5
Wang P, Wei Y, Fan Y, Liu Q, Wei W, Yang C, Zhang L, Zhao G, Yue J, Yan X, Zhou Z (2015) Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng. https://doi.org/10.1016/j.ymben.2015.03.003
Wei W, Wang P, Wei Y, Liu Q, Yang C, Zhao G, Yue J, Yan X, Zhou Z (2015) Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8(9):1412–1424. https://doi.org/10.1016/j.molp.2015.05.010
Wen Z, Zhang ZM, Zhong L, Fan J, Li M, Ma Y, Zhou Y, Zhang W, Guo B, Chen B (2021) Directed evolution of a plant glycosyltransferase for chemo- and regioselective glycosylation of pharmaceutically significant flavonoids. ACS Catal 24:14781–14790. https://doi.org/10.1021/acscatal.1c04191
Xiang L, Zhu S, Zhao T, Zhang M, Liu W, Chen M, Lan X, Liao Z (2015) Enhancement of artemisinin content and relative expression of genes of artemisinin biosynthesis in Artemisia annua by exogenous MeJA treatment. Plant Growth Regul 75(2):435–441. https://doi.org/10.1007/s10725-014-0004-z
Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24(6):770–773. https://doi.org/10.1038/cr.2014.28
Yang C, Li C, Wei W, Wei Y, Liu Q, Zhao G, Yue J, Yan X, Wang P, Zhou Z (2020) The unprecedented diversity of UGT94-family UDP-glycosyltransferases in Panax plants and their contribution to ginsenoside biosynthesis. Sci Rep 10(1):15394. https://doi.org/10.1038/s41598-020-72278-y
Yang Z, Li X, Yang L, Peng S, Song W, Lin Y, Xiang G, Li Y, Ye S, Ma C, Miao J, Zhang G, Chen W, Yang S, Dong Y (2023) Comparative genomics reveals the diversification of triterpenoid biosynthesis and origin of ocotillol-type triterpenes in Panax. Plant Commun. 16:100591. https://doi.org/10.1016/j.xplc.2023.100591
Yurry Um, Yi L, Seong-Cheol K, Yeon-Ju J, Geum-Soog K (2017) Expression analysis of ginsenoside biosynthesis-related genes in methyl jasmonate-treated adventitious roots of Panax ginseng via DNA microarray analysis. Hortic Environ Biotechnol 58(4):376–383. https://doi.org/10.1007/s13580-017-0041-4
Zhang L, Ren S, Liu X, Liu X, Guo F, Sun W, Feng X, Li C (2020) Mining of UDP-glucosyltransferases in licorice for controllable glycosylation of pentacyclic triterpenoids. Biotechnol Bioeng 117(12):3651–3663. https://doi.org/10.1002/bit.27518
Zhou P, Yang J, Zhu J, He S, Zhang W, Yu R, Zi J, Song L, Huang X (2015) Effects of β-cyclodextrin and methyl jasmonate on the production of vindoline, catharanthine, and ajmalicine in Catharanthus roseus cambial meristematic cell cultures. Appl Microbiol Biotechnol 99(17):7035–7045. https://doi.org/10.1007/s00253-015-6651-9
Zhu S, Zou K, Fushimi H, Cai S, Komatsu K (2004) Comparative study on triterpene saponins of Ginseng drugs. Planta Med 70(7):666–677. https://doi.org/10.1055/s-2004-827192
Acknowledgements
This work was supported by National Natural Science Foundation of China under Grant (81860680), Major Science and Technology Programs in Yunnan Province (2019ZF011-1), Major Science and Technology Programs in Yunnan Province (202102AA310048), the Science and Technology Innovation Team of Yunnan (202105AE160011), the General Project for Basic Research in Yunnan (Grant No. 202201AT070266).
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Peng, S., Li, X., Jiang, W. et al. Identification of two key UDP-glycosyltransferases responsible for the ocotillol-type ginsenoside majonside-R2 biosynthesis in Panax vietnamensis var. fuscidiscus. Planta 257, 119 (2023). https://doi.org/10.1007/s00425-023-04143-x
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DOI: https://doi.org/10.1007/s00425-023-04143-x