Innovation in malaria medicines from plants: Combinatorial plant metabolic engineering brings solution for malaria
Introduction
Fig. 1. Postdoc and student sampling Artemisia annua growing in Wageningen. |
Secondary metabolites are compounds in plants that are supposed to be of secondary importance for growth and survival of plants. However it is becoming clear that many of these secondary metabolites play a pivotal role for survival of plants. They are for example essential for the attraction of pollinators, the attraction of symbiotic organisms such as mycorrhizal fungi and defense against pathogens and insects. Secondary metabolites may however also be of prime importance to humans. Artemisinin, for example, is a sesquiterpene that is produced in only one plant species, Artemisia annua, and is the most important anti-malaria medicine we currently have (Fig. 1). In an innovative new approach, the group of prof. Harro Bouwmeester from the Laboratory of Plant Physiology and Plant Research International of Wageningen UR will combine – through genetic modification - the artemisinin biosynthetic potential of A. annua with the efficient enzymes and highly industrialized culture of inuline chicory to optimise the production of the anti-malarial artemisinin.
Malaria and ACTs
According to the WHO some 300 to 500 million malaria cases per year are reported worldwide. Each year this results in the death of 1.5 to 2 million people, of which 90% occur in Africa. Rapid treatment with an ACT could save many lives. ACTs (Artemisinin based Combination Therapies) are the latest generation and most effective antimalaria treatment according to the WHO (World Health Organization of the UN). Artemisinin (Fig. 2), the basic raw material used in all ACTs, however, is relatively expensive as its only source is a plant, Artemisia annua, growing mainly in China and Vietnam and only producing low yields. Therefore, an ACT these days easily costs ten times more than a treatment with e.g. chloroquine. This is too expensive for most African patients and this can only be solved if the price of the ACTs, and thus particularly the price of artemisinin, strongly decreases.
Biotechnological production of artemisinin
Fig. 2. Biosynthetic routes of bitter sesquiterpene lactones in chicory and artemisinin in Artemisia annua. |
In earlier research in Wageningen the complete biosynthetic pathway of artemisinin was resolved (Fig. 2). In addition, the Wageningen scientists, headed by Prof. Harro Bouwmeester and Dr. Maurice Franssen of the Laboratory for Organic Chemistry, could demonstrate that chicory enzymes – normally involved in the biosynthesis of the bitter sesquiterpene lactones in chicory (Cichorium intybus) - are capable of performing reactions required for the biosynthesis of artemisinin. Via a diversion of the biosynthesis of bitter compounds we will now, in collaboration with Dafra Pharma NV, produce the chemical precursor for artemisinin (dihydroartemisinic acid) in the roots of chicory. The group of Prof. Bouwmeester has shown in a wide range of plant species that diversion of the biosynthesis of terpenes can be carried out very efficiently.
Why chicory?
Although there is only one species name, Cichorium intybus, chicory comes in many variations. Best known is witloof chicory, a slightly bitter vegetable that is eaten particularly in countries such as the Netherlands, Belgium and France. However, other chicory varieties were selected particularly for their high inulin producing taproot (Fig. 3). Inulin is a long chain carbohydrate which is used in food (inulin is a dietary fiber) as well as non-food applications. Because of this, inulin chicory is a well-established industrial crop and the entire chain of large-scale agricultural production, including extraction, is already operational (Fig. 3). Considering this and the superb enzymes that are present in chicory, we are convinced that inulin chicory can serve as the perfect artemisinin production platform.
Conclusion
The research will result in the cost-effective, large-scale production of artemisinin which should lead to cheap, but high-quality, effective and safe anti-malaria treatments (ACTs).
Publications
1) Bouwmeester, H.J., T.E. Wallaart, M.H.A. Janssen, B. van Loo, B.J.M. Jansen, M.A. Posthumus, C.O. Schmidt, J-W. de Kraker, W.A. König and M.C.R. Franssen, 1999. Partial purification and characterization of amorpha-4,11-diene synthase. The sesquiterpene synthase catalyzing the first probable step in the biosynthesis of artemisinin. Phytochemistry 52: 843-854.
2) Mercke, P., M. Bengtsson, H.J. Bouwmeester, M.A. Posthumus and P.E. Brodelius, 2000. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Archives of Biochemistry and Biophysics 381(2): 173-180
3) Wallaart, T.E., H.J. Bouwmeester, J. Hille, L. Poppinga and N.C.A. Maijers, 2001. Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 212(3): 460-465.
4) de Kraker, JW, Schurink, M, Franssen, MCR, de Groot, Ae, Bouwmeester, HJ, 2003. Hydroxylation of sesquiterpenes by enzymes from chicory (Cichorium intybus L.) roots.Tetrahedron 59: 409-418
5) Bertea, C.M., J. R. Freije, H. van der Woude, F. W. A. Verstappen, L. Perk, V. Marquez, J-W De Kraker, M. A. Posthumus, B. J. M. Jansen, Ae. de Groot, M. C. R. Franssen and H. J. Bouwmeester, 2005. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua L. Planta Medica 71: 40-47
6) Bouwmeester, H.J., C.M. Bertea, J-W. de Kraker, F.W.A. Verstappen and M.C.R. Franssen, 2006. Research to improve artemisinin production for use in the preparation of anti-malarial drugs, In: R.J. Bogers, L.E. Craker and D. Langer (eds) Medicinal and Aromatic Plants, Frontis Workshop 17-20 April 2005, Wageningen, the Netherlands, Kluwer, pp 275-290.
7) Lommen, W., E. Schenk, F.W.A. Verstappen and H.J. Bouwmeester, 2006. Trichome dynamics and artemisinin accumulation during development and senescence of Artemisia annua L. leaves. Planta Medica 72: 336-345
8) Bertea, C.M., A. Voster, F.W.A. Verstappen, M. Maffei, J. Beekwilder, H.J. Bouwmeester, 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
9) W. J. M. Lommen, S. Elzinga, H. J. Bouwmeester and F. W. A. Verstappen, 2007. Artemisinin and sesquiterpene precursors in dead and green leaves of Artemisia annua L. crops. Planta Medica 73(10) 1133 – 1139
10) Lommen, W.J.M., H.J. Bouwmeester, E. Schenk, F.W.A. Verstappen, S. Elzinga, 2007. Processes determining and limiting the production of secondary metabolites during crop growth, using the antimalarial artemisinin produced in Artemisia annua as an example. Acta Horticulturae, in press
Patents
1) Wallaart, T.E. and H.J. Bouwmeester, 1998. DNA encoding amorpha-4,11-diene synthase. Patent PCT/EP99/06302
2) Bouwmeester, H.J., M.C.R. Franssen, J-W. de Kraker, M. Schurink and R. Bino, 2001. Plant enzymes for bioconversion. PCT WO 03/025193 A1
For more information: prof. dr Harro Bouwmeester, Laboratory for Plant Physiology and Plant Research International, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands, tel +31 317 475882, e-mail: harro.bouwmeester@wur.nl