The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g？1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
Zhang S, Won YK, Ong CN, Shen HM (2005) Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms. Curr Med Chem Anticancer Agents 5: 239–249.
Koo TH, Lee J-H, Park YJ, Hong Y-S, Kim HS, et al. (2001) A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-κB by targeting IκB phosphorylation. Planta Med 67: 103–107.
de Kraker JW, Franssen MCR, Joerink M, de Groot A, Bouwmeester HJ (2002) Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome P450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory. Plant Physiology 129: 257–268.
Seto M, Miyase T, Umehara K, Ueno A, Hirano Y, et al. (1988) Sesquiterpene lactones from Cichorium endivia L. and C. intybus L. and cytotoxic activity. Chem Pharm Bull (Tokyo) 36: 2423–2429.
Van Beek TA, Maas P, King BM, Leclercq E, Voragen AGJ, et al. (1990) Bitter sesquiterpene lactones from chicory roots. Journal of Agricultural and Food Chemistry 38: 1035–1038.
Mori H, Kawamori T, Tanaka T, Ohnishi M, Yamahara J (1994) Chemopreventive effect of costunolide, a constituent of oriental medicine, on azoxymethane-induced intestinal carcinogenesis in rats. Cancer Letters 83: 171–175.
Chen H-C, Chou C-K, Lee S-D, Wang J-C, Yeh S-F (1995) Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells. Antiviral Research 27: 99–109.
Barrero AF, Oltra JE, álvarez M, Raslan DS, Saúde DA, et al. (2000) New sources and antifungal activity of sesquiterpene lactones. Fitoterapia 71: 60–64.
Taniguchi M, Kataoka T, Suzuki H, Uramoto M, Ando M, et al. (1995) Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T-Lymphocytes. Bioscience Biotechnology and Biochemistry 59: 2064–2067.
Srivastava SK, Abraham A, Bhat B, Jaggi M, Singh AT, et al. (2006) Synthesis of 13-amino costunolide derivatives as anticancer agents. Bioorganic & Medicinal Chemistry Letters 16: 4195–4199.
Zhang DL, Qiu L, Jin XQ, Guo ZH, Guo CB (2009) Nuclear factor-kappa B inhibition by parthenolide potentiates the efficacy of taxol in non-small cell lung cancer In vitro and In vivo. Molecular Cancer Research 7: 1139–1149.
Bedoya LM, Abad MJ, Bermejo P (2008) The role of parthenolide in intracellular signalling processes: Review of current knowledge. Current Signal Transduction Therapy 3: 82–87.
de Kraker JW, Franssen MCR, Dalm MCF, de Groot A, Bouwmeester HJ (2001) Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene A hydroxylase and NADP(+)-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis. Plant Physiology 125: 1930–1940.
de Kraker JW, Franssen MCR, de Groot A, Konig WA, Bouwmeester HJ (1998) (+)-Germacrene A biosynthesis - The committed step in the biosynthesis of bitter sesquiterpene lactones in chicory. Plant Physiology 117: 1381–1392.
Bouwmeester HJ, Kodde J, Verstappen FWA, Altug IG, de Kraker JW, et al. (2002) Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiology 129: 134–144.
Bertea CM, Voster A, Verstappen FWA, Maffei M, Beekwilder J, et al. (2006) Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Archives of Biochemistry and Biophysics 448: 3–12.
Majdi M, Liu Q, Karimzadeh G, Malboobi MA, Beekwilder J, et al. (2011) Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.). Phytochemistry. in press. doi:10.1016/j.phytochem.2011.1004.1021.
Nguyen DT, Gopfert JC, Ikezawa N, Macnevin G, Kathiresan M, et al. (2010) Biochemical conservation and evolution of germacrene A oxidase in asteraceae. J Biol Chem 285: 16588–16598.
van Herpen TWJM, Cankar K, Nogueira M, Bosch D, Bouwmeester HJ, et al. (2010) Nicotiana benthamiana as a production platform for Artemisinin precursors. PLoS One 5:
Kappers IF, Aharoni A, van Herpen TWJM, Luckerhoff LLP, Dicke M, et al. (2005) Genetic engineering of terpenoid metabolism attracts, bodyguards to Arabidopsis. Science 309: 2070–2072.
Tikunov Y, Lommen A, de Vos CHR, Verhoeven HA, Bino RJ, et al. (2005) A novel approach for nontargeted data analysis for Mmetabolomics. Large-scale profiling of tomato fruit volatiles. Plant Physiology 139: 1125–1137.
Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943.
Tremblay R, Wang D, Jevnikar AM, Ma SW (2010) Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnology Advances 28: 214–221.
Marrs KA (1996) The functions and regulation of glutathione s-transferases in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47: 127–158.
Dean JD, Goodwin PH, Hsiang T (2005) Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance. Journal of Experimental Botany 56: 1525–1533.
Marrs KA, Alfenito MR, Lloyd AM, Walbot V (1995) A glutathione-s-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature 375: 397–400.
Ohkama-Ohtsu N, Zhao P, Xiang CB, Oliver DJ (2007) Glutathione conjugates in the vacuole are degraded by gamma-glutamyl transpeptidase GGT3 in Arabidopsis. Plant Journal 49: 878–888.
Shimizu H, Araki K, Shioya S, Suga K (1991) Optimal production of glutathione by controlling the specific growth-rate of yeast in fed-batch culture. Biotechnology and Bioengineering 38: 196–205.
Choi JH, Ha J, Park JH, Lee JY, Lee YS, et al. (2002) Costunolide triggers apoptosis in human leukemia U937 cells by depleting intracellular thiols. Japanese Journal of Cancer Research 93: 1327–1333.
Park HJ, Jung HJ, Lee KT, Choi J (2006) Biological characterization of the chemical structures of naturally occurring substances with cytotoxicity. Nat Prod Sci, Korea 12: 175–192.
Ma X-C, Zheng J, Guo D-A (2007) Microbial transformation of dehydrocostuslactone and costunolide by Mucor polymorphosporus and Aspergillus candidus. Enzyme and Microbial Technology 40: 1013–1019.
Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plant P450s in optimized redox environments. Cytochrome P450 Pt B 272: 51–64.
Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-Cytochrome P450 reductases with P450 CYP73A5. Journal of Biological Chemistry 272: 19176–19186.
Outchkourov NS, Peters J, de Jong J, Rademakers W, Jongsma MA (2003) The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 216: 1003–1012.
Vanengelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, et al. (1995) Pbinplus - an improved plant transformation vector based on Pbin19. Transgenic Research 4: 288–290.
De Vos RCH, Moco S, Lommen A, Keurentjes JJB, Bino RJ, et al. (2007) Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nature Protocols 2: 778–791.
Kohlen W, Charnikhova T, Liu Q, Bours R, Domagalska MA, et al. (2010) Strigolactones are transported through the xylem and play a key role in shoot architectural response to phosphate deficiency in non-AM host Arabidopsis thaliana. Plant Physiol.
Yang T, Stoopen G, Yalpani N, Vervoort J, de Vos R, et al. (2011) Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns. Metabolic Engineering.
Moco S, Bino RJ, Vorst O, Verhoeven HA, de Groot J, et al. (2006) A liquid chromatography-mass spectrometry-based metabolome database for tomato. Plant Physiology 141: 1205–1218.
Ikezawa N, Goepfert JC, Nguyen DT, Kim S-U, O'Maille PE, et al. (2011) Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereo-selective hydroxylations in sesquiterpene lactone metabolism. Journal of Biological Chemistry.
