Artemisinin biosynthesis in growing plants of Artemisia annua.A 13 CO 2 study Nicholas Schramek b,1 , Huahong Wang a,1 , Werner Römisch-Margl b , Birgit Keil b , Tanja Radykewicz b , Bernhard Winzenhörlein c , Ludger Beerhues d , Adelbert Bacher b , Felix Rohdich b , Jonathan Gershenzon e , Benye Liu a, * , Wolfgang Eisenreich b, * a Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Nanxincun 20, Haidian District, 100093 Beijing, China b Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany c Amt für Grünordnung, Naturschutz und Friedhofswesen der Stadt Augsburg, Botanischer Garten, Dr.-Ziegenspeck-Weg 10, D-86161 Augsburg, Germany d Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany e Department of Biochemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany article info Article history: Received 24 July 2009 Received in revised form 8 October 2009 Available online 22 November 2009 Dedicated to Prof. Hanns-Ludwig Schmidt in honour of his 80th birthday. Keywords: Terpene Biosynthesis Mevalonate 1-Deoxyxylulose phosphate Artemisinin abstract Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate path- way. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of 13 CO 2 for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by 13 C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosyn- thesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Malaria is a leading factor of global morbidity and mortality with an estimated number of 400 million infections and 1–3 mil- lion deaths per year. Approximately 40% of the world population live in areas of active malaria transmission. The disease symptoms are most severe in young children and pregnant women. The emer- gence of resistant Plasmodium strains has made chloroquine, the traditional antimalarial drug, virtually ineffective in most parts of the world (Rathore et al., 2005). Artemisinin (also called Qinghaosu) is a sesquiterpene lactone endoperoxide (11, Fig. 1) that was isolated more than 30 years ago from Artemisia annua by Chinese scientists searching for novel antimalarial drugs (Liu et al., 1979). Artemisinin and some of its derivatives have rapidly become the most important agents in the treatment of malaria, particularly in the form of artemisinin- based combination therapies (ACTs) which are advocated by the WHO in order to reduce the odds of resistance development (Duffy and Mutabingwa, 2006; White, 2008). Currently, artemisinin is the only available drug that is globally effective against malarial parasite. It is characterized by rapid ther- apeutic action and low toxicity levels. Artemisinin inactivates or kills gametocytes of Plasmodium spp. by inhibition of the sarco/ endoplasmic reticulum Ca 2+ ATPase (SERCA) after activation by iron ions (Eckstein-Ludwig et al., 2003). Besides the antimalarial activity, artemisinins have been reported to possess antiviral (Romero et al., 2006), anticancer (Efferth, 2006), and antischistos- omal activity (Utzinger et al., 2007). An estimated number of 400–600 million therapeutic doses of artemisinin will be required for ACT per year, whereas less than 100 million doses per year are presently available. A. annua is the only commercial source for artemisinin. This supply could be im- proved by metabolic engineering of the plant in order to obtain higher yields. However, knowledge about the biosynthetic path- way in growing plants of A. annua is required for this approach. The first committed step in the biosynthesis of artemisinin in A. annua is reported to be the cyclization of (E,E)-farnesyl diphos- phate (1, FPP, Fig. 1)(Kim et al., 2006) catalyzed by amorpha- 4,11-diene synthase for which corresponding cDNAs have been cloned independently by several groups (Chang et al., 2000; 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.10.015 * Corresponding authors. Tel.: +86 10 62836239; fax: +86 10 82591016 (B. Liu), tel.: +49 89 289 13336; fax: +49 89 289 13363 (W. Eisenreich). E-mail addresses: benyel@ibcas.ac.cn (B. Liu), wolfgang.eisenreich@ch.tum.de (W. Eisenreich). 1 The two authors contributed equally to the work. Phytochemistry 71 (2010) 179–187 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem