Abstract
Artemisinin is a sesquiterpene lactone endoperoxide with potent antimalarial properties, recommended by the World Health Organization for the treatment of malaria in artemisinin combination therapies (ACTs). It is extracted from the plant Artemisia annua, but its supplies are limited and its price is volatile. In order to increase supply and stabilize the price of artemisinin, a semisynthesis has been developed, whereby an artemisinin precursor (amorpha-4,11-diene) is produced in microbes and the isolated precursor converted chemically to artemisinin. Escherichia coli has been engineered to produce amorpha-4,11-diene by the expression of a heterologous mevalonate pathway along with amorpha-4,11-diene synthase (ADS) from A. annua. Development of the E. coli platform to increase production of amorpha-4,11-diene from 24 mg/L to >25 g/L is described. ADS has also been expressed in the yeast model system Saccharomyces cerevisiae which, following manipulation of the mevalonate pathway, produced 150 mg/L of amorpha-4,11-diene. The cDNAs encoding the cytochrome P450 that oxidizes amorpha-4,11-diene to artemisinic acid, CYP71AV1, and its cognate reductase were isolated from A. annua and expressed in amorpha-4,11-diene-producing E. coli and yeast, leading to the production of >1 g/L artemisinic acid from both organisms. A route for the chemical conversion of artemisinic acid to artemisinin is described. Production of semisynthetic artemisinin may lead to the development of a second source of the drug for incorporation into ACTs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410
Anthony JR, Anthony LC, Nowroozi F et al (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-Âdiene. Metab Eng 11(1):13–19
Bertea CM, Freije JR, van der Woude H et al (2005) Idenepsication of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med 71(1):40–47
Bloland P (2001) Drug resistance in malaria. World Health Organization, Geneva
Bohme K, Brauer H-D (1992) Generation of singlet oxygen from hydrogen peroxide disproportionation catalyzed by molybdate ions. Inorg Chem 31:3468–3471
Bochar DA, Stauffacher CV, Rodwell VW. (1999) Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mol Genet Metab 66:122–127
Boucher Y, Doolittle WF (2000) The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways. Mol Microbiol 37(4):703–716
Bouwmeester HJ, Wallaart TE, Janssen MH et al (1999) Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 52(5):843–854
Chang MC, Keasling JD (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat Chem Biol 2(12):674–681
Chang MC, Eachus RA, Trieu W, Ro DK, Keasling JD (2007) Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nat Chem Biol 3:274–277
Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68(14):1864–1871
Donald KA, Hampton RY, Fritz IB (1997) Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 63(9):3341–3344
Hale V, Keasling JD, Renninger N, Diagana TT (2007) Microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs. Am J Trop Med Hyg 77(6_Suppl):198–202
Hamann T, Møller BL (2007) Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast. Protein Expr Purif 56(1): 121–127
Hampton R, Dimster-Denk D, Rine J (1996) The biology of HMG-CoA reductase: the pros of contra-regulation. Trends Biochem Sci 21(4):140–145
Haynes RK (2006) From artemisinin to new artemisinin antimalarials: biosynthesis, extraction, old and new derivatives, stereochemistry and medicinal chemistry requirements. Curr Top Med Chem 6(5):509–537
Hedl M, Sutherlin A, Wilding EI et al (2002) Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-Âfunction protein of isopentenyl diphosphate biosynthesis. J Bacteriol 184(8):2116–2122
Hedl M, Tabernero L, Stauffacher CV, Rodwell VW (2004) Class II 3-hydroxy-3-methylglutaryl coenzyme A reductases. J Bacteriol 186(7):1927–1932
Henkel J, Maurer SM (2007) The economics of synthetic biology. Mol Syst Biol 3:117
Hsu E (2006) Reflections on the ‘discovery’ of the Âantimalarial qinghao. Br J Clin Pharmacol 61(6):666–670
Jackson BE, Hart-Wells EA, Matsuda SP (2003) Metabolic engineering to produce sesquiterpenes in yeast. Org Lett 5(10):1629–1632
Kim BJ, Sasaki T (2006) Recent progress in the synthesis of artemisinin and its derivatives. Org Prep Proced Int 38(1):1–80
Kizer L, Pitera DJ, Pfleger BF, Keasling JD (2008) Application of functional genomics to pathway optimization for increased isoprenoid production. Appl Environ Microbiol 74:3229–3241.
Klayman DL (1985) Qinghaosu (artemisinin): an antiÂmalarial drug from China. Science 228(4703):1049–1055
Korenromp E, Miller J, Nahlen B, Wardlaw T, Young M (2005) World malaria report 2005. Roll Back Malaria/World Health Organization/UNICEF, Geneva
Lenihan JR, Tsuruta H, Diola D, Renninger NS, Regentin R (2008) Developing an industrial artemisinic acid fermentation process to support the cost-effective production of anti-malarial artemisinin-based combination therapies (ACTs). Biotechnol Prog 24(5):1025–1032
Li Y, Huang HW, Wu Y-L (2006) Qinghaosu (artemisinin) – a fantastic antimalarial drug from a traditional Chinese medicine. In: Liang X-T, Fang W-S (eds) Medicinal chemistry of bioactive natural products. Wiley, New York, pp 183–256
Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21(7):796–802
McCaskill D, Croteau R (1997) Prospects for the bioengineering of isoprenoid biosynthesis. Adv Biochem Eng Biotechnol 55:107–146
Newman JD, Marshall J, Chang M et al (2006) High-level production of amorpha-4,11-diene in a two-phase Âpartitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol Bioeng 95(4):684–691
Njau JD, Goodman CA, Kachur SP et al (2008) The costs of introducing artemisinin-based combination therapy: evidence from district-wide implementation in rural Tanzania. Malar J 7(1):4
Olumese P (2006) Guidelines for the treatment of malaria, 2nd edn. World Health Organization, Geneva
Paradise EM, Kirby J, Chan R, Keasling JD (2008) Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by downregulating squalene synthase. Biotechnol Bioeng 100(2):371–378
Pfleger BF, Pitera DJ, Smolke CD, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24:1027–1032
Pfleger BF, Pitera DJ, Newman JD, Martin VJ, Keasling JD (2007) Microbial sensors for small molecules: development of a mevalonate biosensor. Metab Eng 9:30–38
Pitera DJ, Paddon CJ, Newman JD, Keasling JD (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9(2):193–207
Reiling KK, Renninger NS, McPhee DJ, Fisher KJ, Ockey DA (2006) Conversion of amorpha-4,11-diene to artemisinin and artemisinin precursors. WIPO (http://www.wipo.int/patentscope/en/) WO/2006/128126. Amyris Biotechnologies, Inc., New York
Ro DK, Paradise EM, Ouellet M et al (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440(7086):940–943
Sy L-K, Brown GD (2002) The role of the 12-carboxylic group in the spontaneous autoxidation of dihydroartemisinic acid. Tetrahedron 58(5):909–923
Tabata K, Hashimoto S (2004) Production of mevalonate by a metabolically-engineered Escherichia coli. Biotechnol Lett 26(19):1487–1491
Teoh KH, Polichuk DR, Reed DW, Nowak G, Covello PS (2006) Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett 580(5):1411–1416
Tsuruta H, Paddon CJ, Eng D, Lenihan JR et al (2009) High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One 4:e4489
van Agtmael MA, Eggelte TA, van Boxtel CJ (1999) Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication. Trends Pharmacol Sci 20(5):199–205
Vik A, Rine J (2001) Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cerevisiae. Mol Cell Biol 21(19):6395–6405
Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1(6): REVIEWS3003
White NJ (2008) Qinghaosu (artemisinin): the price of success. Science 320(5874):330–334
Zhang Y, Teoh KH, Reed DW, Maes L, Goossens A, Olson DJ, Ross AR, Covello PS. (2008) The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem 283: 21501–21508
Zeng Q, Qiu F, Yuan L (2007) Production of artemisinin by genetically-modified microbes. Biotechnol Lett 30(4):581–592
Acknowledgements
We thank Jay Keasling and his lab members for many productive conversations. This research was conducted under the sponsorship of the Institute for OneWorld Health through the generous support of the Bill & Melinda Gates Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this chapter
Cite this chapter
Paddon, C.J. et al. (2012). Microbially Derived Semisynthetic Artemisinin. In: Bach, T., Rohmer, M. (eds) Isoprenoid Synthesis in Plants and Microorganisms. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-4063-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-4614-4063-5_7
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4614-4062-8
Online ISBN: 978-1-4614-4063-5
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)