Plant Cell Reports

, Volume 26, Issue 12, pp 2129–2136 | Cite as

Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways

Physiology and Biochemistry


The potent antimalarial sesquiterpene lactone, artemisinin, is produced in low quantities by the plant Artemisia annua L. The source and regulation of the isopentenyl diphosphate (IPP) used in the biosynthesis of artemisinin has not been completely characterized. Terpenoid biosynthesis occurs in plants via two IPP-generating pathways: the mevalonate pathway in the cytosol, and the non-mevalonate pathway in plastids. Using inhibitors specific to each pathway, it is possible to resolve which supplies the IPP precursor to the end product. Here, we show the effects of inhibition on the two pathways leading to IPP for artemisinin production in plants. We grew young (7–14 days post cotyledon) plants in liquid culture, and added mevinolin to the medium to inhibit the mevalonate pathway, or fosmidomycin to inhibit the non-mevalonate pathway. Artemisinin levels were measured after 7–14 days incubation, and production was significantly reduced by each inhibitor compared to controls, thus, it appears that IPP from both pathways is used in artemisinin production. Also when grown in miconazole, an inhibitor of sterol biosynthesis, there was a significant increase in artemisinin compared to controls suggesting that carbon was shifted from sterols into sesquiterpenes. Collectively these results indicate that artemisinin is probably biosynthesized from IPP pools from both the plastid and the cytosol, and that carbon from competing pathways can be channeled toward sesquiterpenes. This information will help advance our understanding of the regulation of in planta production of artemisinin.


Artemisinin Mevalonate pathway Nonmevalonate pathway DMSO 



Thanks to Brandon Matthews for some preliminary work. We also appreciate assistance with statistics by Professor Liz Ryder of WPI, and the critical reviews provided by Professors Kris Wobbe at WPI, and Fabricio Medina-Bolivar at the Arkansas Bioscience Institute. Financial support for this work was gratefully received from NIH 1R15GM 069562-01.


  1. Adam K, Zapp J (1998) Biosynthesis of the isoprene units of chamomile sesquiterpenes. Phytochem Anal 48:953–959CrossRefGoogle Scholar
  2. Adam K, Thiel R, Zapp J, Becker H (1998) Involvement of the mevalonic acid pathway and the glyceraldehyde-pyruvate pathway in terpenoid biosynthesis of the liverworts Ricciocarpos natans and Conocephalum conicum. Arch Biochem Biophys 354:181–187PubMedCrossRefGoogle Scholar
  3. Arigoni D, Eisenreich W, Latzel C, Sagner S, Radykewicz T, Zenk MH, Bacher A (1999) Dimethylallyl pyrophosphate is not the committee precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants. Proc Natl Acad Sci USA 96:1309–1314PubMedCrossRefGoogle Scholar
  4. Bach TJ, Benveniste P (1997) Cloning of cDNAs or genes encoding enzymes of sterol biosynthesis from plants and other eukaryotes: heterologous expression and complementation analysis of mutations for functional characterization. Prog Lipid Res 36:197-226PubMedCrossRefGoogle Scholar
  5. Bach TJ, Lichtenthaler HK (1983) Inhibition by mevinolin of plant growth, sterol formation and pigment accumulation. Physiol Plant 59:50–60CrossRefGoogle Scholar
  6. Bajaj YPS, Rathore VS, Wittwer SH, Adams MW (1970) Effect of dimethyl sulfoxide on zinc65 uptake, respiration, and RNA and protein metabolism in bean (Phaseolus vulgaris) tissues. Am J Bot 57:794–799CrossRefGoogle Scholar
  7. Bartram S, Jux A, Gleixner G, Boland W (2006) Dynamic pathway allocation in early terpenoid biosynthesis of stress-induced lima bean leaves. Phytochem 67:1661–1672CrossRefGoogle Scholar
  8. Carswell GK, Johnson CM, Shillito RD, Harms CT (1989) O-acetyl-salicylic acid promotes colony formation from protoplasts of an elite maize inbred. Plant Cell Rep 8:282–284CrossRefGoogle Scholar
  9. Chen Z, Zhang Y (2005) Dimethyl sulfoxide targets phage RNA polymerases to promote transcription. Biochem Biophys Res Commun 333:664–670PubMedCrossRefGoogle Scholar
  10. Dewick PM (2002) The biosynthesis of C5-C25 terpenoid compounds. Nat Prod Rep 19:181–222PubMedCrossRefGoogle Scholar
  11. Dubey VS, Bhalla R, Luthra R (2003) An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants. J Biosci 28:637–646PubMedCrossRefGoogle Scholar
  12. Dudareva N, Andersson S, Orlova I, Gatto N, Reichelt M, Rhodes D, Boland W, Gershenzon J (2005) The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers. Proc Natl Acad Sci USA 102:933–938PubMedCrossRefGoogle Scholar
  13. Ferreira JFS, Janick J (1996) Roots as an enhancing factor for the production of artemisinin in shoot cultures of Artemisia annua. Plant Cell Tissue Organ Cult 44:211–217CrossRefGoogle Scholar
  14. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:148–151CrossRefGoogle Scholar
  15. Hahne G, Hoffmann F (1984) Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly. Proc Natl Acad Sci USA 81:5449–5453PubMedCrossRefGoogle Scholar
  16. Heintze A, Gorlach J, Leuschner C, Hoppe P, Hagelstein P, Schulze-Siebert D, Schultz G (1990) Plastidic isoprenoid synthesis during chloroplast development. Plant Physiol 93:1121-1127PubMedCrossRefGoogle Scholar
  17. Hemmerlin A, Bach TJ (1998) Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells. Plant J 14:65–74PubMedCrossRefGoogle Scholar
  18. Hemmerlin A, Hoeffler JF, Meyer O, Tritsch D, Kagan IA, Grosdemange-Billiard C, Rohmer M, Bach T (2003) Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. J Biol Chem 278:26666–26676PubMedCrossRefGoogle Scholar
  19. Kasahara H, Hanada A, Kuzuyama T, Takagi M, Kamiya Y, Yamaguchi S (2002) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J Biol Chem 277:45188–45194PubMedCrossRefGoogle Scholar
  20. Kim HB, Schaller H, Goh CH, Kwon M, Choe S, An CS, Durst F, Feldmann KA, Feyereisen R (2005) Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity. Plant Physiol 138:2033–2047PubMedCrossRefGoogle Scholar
  21. Kudakasseril GJ, Lam L, Staba EJ 1987 Effect of sterol inhibitors on the incorporation of 14C-isopentenyl pyrophosphate into artemisinin by a cell-free system from Artemisia annua tissue cultures and plants. Planta Med 53:280–284PubMedCrossRefGoogle Scholar
  22. Laule O, Fürholz A, Chang HS, Zhu T, Wang X, Heifetz, Gruissem W, Lange BM (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 100:6866–6871PubMedCrossRefGoogle Scholar
  23. Maier W, Schneider B, Strack D (1998) Biosynthesis of sesquiterpenoid cyclohexenone derivatives in mycorrhizal barley roots proceeds via the glyceraldehyde 3-phosphate/pyruvate pathway. Tetrahedron Lett 39:521–524CrossRefGoogle Scholar
  24. Re EB, Jones D, Learned RM (1995) Co-expression of native and introduced genes reveals cryptic regulation of HMG CoA reductase expression in Arabidopsis. Plant J 7:771–784PubMedCrossRefGoogle Scholar
  25. Rodrígues-Concepción M, Gruissem W (1999) Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation. Plant Physiol 119:41–48CrossRefGoogle Scholar
  26. Rodrígues-Concepción M, Fores O, Martinez-Garcia JF, Gonzalez V, Phillips MA, Ferrer A, Boronat A (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16:144–156CrossRefGoogle Scholar
  27. Rohmer M, Seemann M, Horbach S, Bringermeyer S, Sahm H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118:2654–2566CrossRefGoogle Scholar
  28. Sacchettini JC, Poulter CD (1997) Creating isoprenoid diversity. Science 277:2788–1789CrossRefGoogle Scholar
  29. Santos NC, Figueira-Coelho J, Martins-Silva J, Saldanha C (2003) Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecule aspects. Biochem Pharmacol 65:1035–1041PubMedCrossRefGoogle Scholar
  30. Schwarz KM (1994) Terpen-biosynthese in Ginkgo biloba: Eine Uberraschende geschichte, Ph.D. Thesis, Eidgenossischen Technischen Hochschule, Zurich, SwitzerlandGoogle Scholar
  31. Smith TC, Weathers PJ, Cheetham RD (1997) Effects of gibberellic acid on hairy root cultures of Artemisia annua: growth and artemisinin production. In Vitro Cell Dev Biol Plant 33:75–79CrossRefGoogle Scholar
  32. Steliopoulos P, Wüst M, Adam K-P, Mosandl A (2002) Biosynthesis of the sesquiterpene germacrene D in Solidago cnadensis: 13C and 2H labeling studies. Phytochem Anal 60:13–20CrossRefGoogle Scholar
  33. Thiel R, Adam KP (2002) Incorporation of [1–13C]1-deoxy-d-xylulose into isoprenoids of the liverwort Conocephalum conicum. Phytochem Anal 59:269–274CrossRefGoogle Scholar
  34. Wang Y, Weathers PJ (2007) Sugars proportionately affect artemisinin production. Plant Cell Rep 26:1073–1081PubMedCrossRefGoogle Scholar
  35. Weathers PJ, Cheetham RD, Follansbee E, Teoh K (1994) Artemisinin production by transformed roots of Artemisia annua. Biotechnol Lett 16:1281–1286Google Scholar
  36. Weathers PJ, DeJesus-Gonzalez L, Kim YJ, Souret FF, Towler MJ (2004) Alteration of biomass and artemisinin production in A. annua hairy roots by media sterilization method and sugars. Plant Cell Rep 23:414–418PubMedCrossRefGoogle Scholar
  37. Wu S, Schalk M, Clark A, Miles RB, Coates R, Chappell J (2006) Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotechnol 24:1441–1447PubMedCrossRefGoogle Scholar
  38. Zarn JA, Brüschweiler BJ, Schlatter JR (2003) Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14α-demethylase and aromatase. Environ Health Perspect 111:255–261PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Biology/BiotechnologyWorcester Polytechnic InstituteWorcesterUSA
  2. 2.Arkansas Bioscience InstituteArkansas State UniversityState UniversityUSA

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