CN: 32-1845/R
ISSN: 2095-6975
Cite this paper:
TONG Yu-Ru, ZHANG Yi-Feng, ZHAO Yu-Jun, HU Tian-Yuan, WANG Jia-Dian, HUANG Lu-Qi, GAO Wei. Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii[J]. Chinese Journal of Natural Medicines, 2019, 17(8): 575-584

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii

TONG Yu-Ru1,2,3, ZHANG Yi-Feng3,4, ZHAO Yu-Jun2, HU Tian-Yuan3,4, WANG Jia-Dian3,4, HUANG Lu-Qi1,2, GAO Wei3,4,5
1 School of Pharmacy Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;
2 National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
3 School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China;
4 School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China;
5 Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing 100069, China
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.
Key words:    Overexpression    RNAi    HMGS    Triptolide    Acetyl-CoA   
Received: 2019-04-22   Revised:
PDF (4806 KB) Free
Print this page
Email this article to others
Articles by TONG Yu-Ru
Articles by ZHANG Yi-Feng
Articles by ZHAO Yu-Jun
Articles by HU Tian-Yuan
Articles by WANG Jia-Dian
Articles by HUANG Lu-Qi
Articles by GAO Wei
[1] Goldbach-Mansky R, Wilson M, Fleischmann R, et al. Comparison of Tripterygium wilfordii Hook F versus sulfasalazine in the treatment of rheumatoid arthritis:a randomized trial[J]. Ann Intern Med, 2009, 151(4):229-240, w249-251.
[2] Wang M, Huang J, Fan H, et al. Treatment of rheumatoid arthritis using combination of methotrexate and tripterygium glycosides tablets-A quantitative plasma pharmacochemical and pseudotargeted metabolomic approach[J]. Front Pharmacol, 2018, 9:1051.
[3] He QL, Minn I, Wang Q, et al. Targeted delivery and sus-tained antitumor activity of triptolide through glucose conjugation[J]. Angew Chem Int Ed Engl, 2016, 55(39):12035-12039.
[4] Liu J, Lee J, Salazar Hernandez MA, et al. Treatment of obesity with celastrol[J]. Cell, 2015, 161(5):999-1011.
[5] Hu M, Luo Q, Alitongbieke G, et al. Celastrol-induced Nur77 interaction with TRAF2 alleviates inflammation by promoting mitochondrial ubiquitination and autophagy[J]. Mol Cell, 2017, 66(1):141-153.
[6] Dandawate PR, Subramaniam D, Jensen RA, et al. Tar-geting cancer stem cells and signaling pathways by phyto-chemicals:Novel approach for breast cancer therapy[J]. Semin Cancer Biol, 2016, 40-41:192-208.
[7] Kuzuyama T, Seto H. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis[J]. Proc Jpn Acad Ser B Phys Biol Sci, 2012, 88(3):41-52.
[8] Lombard J, Moreira D. Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life[J]. Mol Biol Evol, 2011, 28(1):87-99.
[9] Puisac B, Marcos-Alcalde I, Hernandez-Marcos M, et al. Human mitochondrial HMG-CoA synthase deficiency:Role of enzyme dimerization surface and characterization of three new patients[J]. Int J Mol Sci, 2018, 19(4):E1010.
[10] Meng X, Song Q, Ye J, et al. Characterization, function, and transcriptional profiling analysis of 3-hydroxy-3-methylglutaryl-CoA synthase gene (GbHMGS1) towards stresses and exogenous hormone treatments in Ginkgo biloba[J]. Molecules, 2017, 22(10):E1706.
[11] Peralta-Yahya PP, Ouellet M, Chan R, et al. Identification and microbial production of a terpene-based advanced biofuel[J]. Nat Commun, 2011, 2:483.
[12] Liu YJ, Zhao YJ, Zhang M, et al. Cloning and characteri-sation of the gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase in Tripterygium wilfordii[J]. Molecules, 2014, 19(12):19696-19707.
[13] Lee AR, Kwon M, Kang MK, et al. Increased sesqui-and triterpene production by co-expression of HMG-CoA reductase and biotin carboxyl carrier protein in tobacco (Nicotiana benthamiana)[J]. Metab Eng, 2019, 52:20-28.
[14] Liao P, Chen X, Wang M, et al. Improved fruit alpha-toco-pherol, carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE1 in transgenic tomato[J]. Plant Biotechnol J, 2018, 16(3):784-796.
[15] Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA[J]. Nature, 2004, 431(7006):343-349.
[16] Miziorko HM, Clinkenbeard KD, Reed WD, et al. 3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation[J]. J Biol Chem, 1975, 250(15):5768-5773.
[17] Tong Y, Su P, Zhao Y, et al. Molecular cloning and characterization of DXS and DXR genes in the terpenoid biosynthetic pathway of Tripterygium wilfordii[J]. Int J Mol Sci, 2015, 16(10):25516-25535.
[18] Zhao Y, Zhang Y, Su P, et al. Genetic transformation sys-tem for woody plant Tripterygium wilfordii and its application to product natural celastrol[J]. Front Plant Sci, 2017, 8:2221.
[19] Tong YR, Su P, Guan HY, et al. Eudesmane-type ses-quiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii[J]. Biochem J, 2018, 475(17):2713-2725.
[20] Boucher Y, Doolittle WF. The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways[J]. Mol Microbiol, 2000, 37(4):703-716.
[21] Kai G, Li S, Wang W, et al. Molecular cloning and ex-pression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Camptotheca acuminata[J]. Rus J Plant Physiol, 2013, 60(1):131-138.
[22] Pojer F, Ferrer JL, Richard SB, et al. Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases[J]. Proc Natl Acad Sci U S A, 2006, 103(31):11491-11496.
[23] Fu Z, Runquist JA, Forouhar F, et al. Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA lyase insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria[J]. J Biol Chem, 2006, 281(11):7526-7532.
[24] Skaff DA, Ramyar KX, McWhorter WJ, et al. Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin[J]. Biochemistry, 2012, 51(23):4713-4722.
[25] Su P, Guan H, Zhao Y, et al. Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii[J]. Plant J, 2018, 93(1):50-65.
[26] Theisen MJ, Misra I, Saadat D, et al. 3-Hydroxy-3-methylglutaryl-CoA synthase intermediate complex observed in "real-time"[J]. Proc Natl Acad Sci U S A, 2004, 101(47):16442-16447.
[27] Steussy CN, Vartia AA, Burgner JW, et al. X-ray crystal structures of HMG-CoA synthase from Enterococcus faecalis and a complex with its second substrate/inhibitor acetoacetyl-CoA[J]. Biochemistry, 2005, 44(43):14256-14267.
[28] Steussy CN, Robison AD, Tetrick AM, et al. A structural limitation on enzyme activity:the case of HMG-CoA synthase[J]. Biochemistry, 2006, 45(48):14407-14414.
[29] Vogeli B, Engilberge S, Girard E, et al. Archaeal acetoace-tyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site[J]. Proc Natl Sci U S A, 2018, 115(13):3380-3385.
[30] Maloney FP, Gerwick L, Gerwick WH, et al. Anatomy of the beta-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate[J]. Proc Natl Acad Sci U S A, 2016, 113(37):10316-10321.
[31] Wang H, Nagegowda DA, Rawat R, et al. Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance[J]. Plant Biotechnol J, 2012, 10(1):31-42.
[32] Mendoza-Poudereux I, Kutzner E, Huber C, et al. Meta-bolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender[J]. Plant Physiol Biochem, 2015, 95:113-120.
[33] Opitz S, Nes WD, Gershenzon J. Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings[J]. Phytochemistry, 2014, 98:110-119.
[34] Hemmerlin A, Hoeffler JF, Meyer O, et al. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells[J]. J Biol Chem, 2003, 278(29):26666-26676.
[35] Weise SE, Li Z, Sutter AE, et al. Measuring dimethylallyl diphosphate available for isoprene synthesis[J]. Anal Biochem, 2013, 435(1):27-34.
[36] Zhang Y, Zhao Y, Wang J, et al. Overexpression and RNA interference of TwDXR regulate the accumulation of terpenoid active ingredients in Tripterygium wilfordii[J]. Biotechnol Lett, 2018, 40(2):419-425.
[37] Su P, Cheng Q, Wang X, et al. Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant, Tripterygium wilfordii, by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry[J]. Biomed Chromatogr, 2014, 28(9):1183-1192.
[38] Wang J, Zhao Y, Zhang Y, et al. Overexpression and RNAi-mediated downregulation of TwIDI regulates triptolide and celastrol accumulation in Tripterygium wilfordii[J]. Gene, 2018, 679:195-201.
[39] Nagegowda DA, Bach TJ, Chye ML. Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1:expression and characterization of recombinant wild-type and mutant enzymes[J]. Biochem J, 2004, 383(Pt. 3):517-527.