Recent developments in synthetic biology, combined with continued progress in systems biology and metabolic engineering, have enabled the engineering of microorganisms to produce heterologous molecules in a manner that was previously unfeasible. The successful synthesis and recent entry of semi-synthetic artemisinin into commercial production is the first demonstration of the potential of synthetic biology for the development and production of pharmaceutical agents. In this Review, we describe the metabolic engineering and synthetic biology approaches that were used to develop this important antimalarial drug precursor. This not only demonstrates the incredible potential of the available technologies but also illuminates how lessons learned from this work could be applied to the production of other pharmaceutical agents.
At a glance
- 2004). Aspirin and Related Drugs (CRC Press,
- Working Knowledge: aspirin. Sci. Am. 280, 108 (1999).
- Untersuchungen über die wasserfreien organischen Säuren. Justus Liebigs Annalen Chemie 87, 57–84 (in German) (1853).
- Qinghaosu (artemisinin): the price of success. Science 320, 330–334 (2008).
A review of the history and properties of artemisinin.
- How Chinese scientists discovered qinghaosu (artemisinin) and developed its derivatives? What are the future perspectives? Med. Trop. 58, 9–12 (1998). &
- World Health Organisation. Guidelines for the treatment of malaria. 2nd edn (WHO, 2010).
- World Health Organisation. WHO informal consultation with manufacturers of artemisinin-based pharmaceutical products in use for the treatment of malaria (WHO, 2007).
- Microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs. Am. J. Trop. Med. Hyg. 77, 198–202 (2007). , , &
- World Health Organisation. World Malaria Report 2012 (WHO, 2012).
- Reflections on the 'discovery' of the antimalarial qinghao. Br. J. Clin. Pharmacol. 61, 666–670 (2006).
- Qinghaosu Antimalarial Coordinating Research Group. Antimalaria Studies on Qinghaosu. Chin. Med. J. 12, 811–816 (1979).
- Qinghaosu (artemisinin): an antimalarial drug from China. Science 228, 1049–1055 (1985).
- [online], (2012). Artemisia Annua, artemisinin, ACTs & malaria control in Africa. Tradition, science and public policy
- Discovery, mechanisms of action and combination therapy of artemisinin. Expert Rev. Anti-Infective Ther. 7, 999–1013 (2009). &
- Impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in Africa: case studies of South Africa, Zambia and Ethiopia. Malar J. 8, S8 (2009). , &
- A major genome region underlying artemisinin resistance in Malaria. Science 336, 79–82 (2012). et al.
- Artemisinin resistance: current status and scenarios for containment. Nature Rev. Microbiol. 8, 272–280 (2010). et al.
- Artemisinin-resistant Malaria: research challenges, opportunities, and public health implications. Am. J. Trop. Med. Hyg. 87, 231–241 (2012). et al.
- Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia. Proc. Natl Acad. Sci. 110, 240–245 (2013). et al.
- Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nature Genet. 45, 648–655 (2013). et al.
- A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014).
This paper reports the identification of a molecular marker for artemisinin-resistant malaria, which will be important for the tracking and elimination of artemisinin-resistant parasites.
- Stabilizing supply of artemisinin and artemisinin-based combination therapy in an era of wide-spread scale-up. Malar J. 11, 399 (2012). &
- Dahlberg Global Development Advisors. Independent Mid-Term review of the assured Artemisinin Supply System (A2S2) Project. Geneva: UNITAID [online], (2012).
- Assured Artemisinin Supply System. Artemisinin imports into India [online], (updated 13 January 2014).
- WHO Health Financing. Per capita total expenditure on health at average exchange rate (US $) [online], (2011).
- The World Bank. Health expenditure per capita (current US$). [online] (2013).
- Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–47 (2005). et al.
- The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao). Molecules 15, 7603–7698 (2010).
- Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 52, 843–854 (1999). et al.
- Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68, 1864–1871 (2007). , , , &
- 91–106 (Springer, 2013). et al. In Isoprenoid Synthesis in Plants and Microorganisms (eds Bach, T. J. & Rohmer, M.)
- The molecular cloning of artemisinic aldehyde Δ11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J. Biol. Chem. 283, 21501–21508 (2008). et al.
- Methylerythritol phosphate pathway of isoprenoid biosynthesis. Annu. Rev. Biochem. 82, 497–530 (2013). , , , &
- Mono and diterpene production in Escherichia coli. Biotechnol. Bioeng. 87, 200–212 (2004). et al.
- Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotech. 21, 796–802 (2003).
This paper provides a description of the initial amorphadiene production pathway in E. coli.
, , , &
- The in vivo synthesis of plant sesquiterpenes by Escherichia coli. Biotechnol. Bioeng. 75, 497–503 (2001). , &
- High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol. Bioeng. 95, 684–691 (2006). et al.
- Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9, 193–207 (2007). , , &
- Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nature Biotech. 24, 1027–1032 (2006). , , &
- Microbial sensors for small molecules: development of a mevalonate biosensor. Metab. Eng. 9, 30–38 (2007). , , , &
- Application of functional genomics to pathway optimization for increased isoprenoid production. Appl. Environ. Microbiol. 74, 3229–3241 (2008). , , &
- Production of mevalonate by a metabolically-engineered Escherichia coli. Biotechnol. Lett. 26, 1487–1491 (2004). &
- Class II 3-hydroxy-3-methylglutaryl coenzyme A reductases. J. Bacteriol. 186, 1927–1932 (2004). , , &
- High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS ONE 4, e4489 (2009).
This study reports strain engineering and fermentation development, which culminated in the production of 25 g per L amorphadiene in E. coli.
- A simple conversion of artemisinic acid into artemisinin. J. Nature Prod. 52, 1183–1185 (1989). &
- 1991). & Simple conversion of artemisinic acid into artemisinin. US Patent 4992561 (
- Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).
This paper describes the isolation of CYP71AV1 — the cytochrome P450 enzyme that oxidizes amorphadiene — and provides the first demonstration of artemisinic acid production in S. cerevisiae.
- Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nature Chem. Biol. 3, 274–277 (2007). , , , &
- Developing an industrial artemisinic acid fermentation process to support the cost-effective production of antimalarial artemisinin-based combination therapies. Biotechnol. Prog. 24, 1026–1032 (2008). , , , &
- Genealogy of principal strains of the yeast genetic stock center. Genetics 113, 35–43 (1986). &
- Life with 6000 genes. Science 274, 563–567 (1996). et al.
- Four linked genes participate in controlling sporulation efficiency in budding yeast. PLoS Genet. 2, e195 (2006). et al.
- An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26, 706–714 (2000). et al.
- Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc. Natl Acad. Sci. USA 109, E111–E118 (2012).
This paper describes strain engineering and fermentation development, which enabled the production of 40 g per L amorphadiene in S. cerevisiae, followed by the chemical conversion of amorphadiene to dihydroartemisinic acid.
- High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).
This paper describes strain engineering and fermentation development — which enabled the production of 25 g per L artemisinic acid in S. cerevisiae — and an efficient, non-photochemical conversion to artemisinin.
- Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. BMC Biotechnol. 8, 83 (2008). et al.
- Mechanisms that regulate production of reactive oxygen species by cytochrome P450. Toxicol. Appl. Pharmacol. 199, 316–331 (2004). , &
- Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase:cytochrome P-450 interaction. J. Biol. Chem. 251, 4010–4016 (1976). , , , &
- The many roles of cytochrome b5. Pharmacol. Ther. 97, 139–152 (2003). &
- Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4. J. Biol. Chem. 282, 29766–29776 (2007). , &
- Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany 87, 635–642 (2009).
This study reports the identification of the A. annua aldehyde dehydrogenase, which is required for high-level production of artemisinic acid in S. cerevisiae.
, , &
- Sanofi. Prix Potier 2012 (in French) [online], (2012).
- WHO Prequalificaion of Medicines Programme. Acceptance of non-plant-derived-artemisinin offers potential to increase access to malaria treatment [online], (2013).
- Isoprenoid pathway optimization for taxol precursor overproduction in E. coli. Science 330, 70–74 (2010). et al.
- Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab. Eng. 10, 201–206 (2008). , &
- Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nature Chem. Biol. 4, 564–573 (2008). &
- Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl Acad. Sci. USA 105, 7393–7398 (2008). et al.
- Recent progress in the metabolic engineering of alkaloids in plant systems. Curr. Opin. Biotechnol. 24, 354–365 (2013). , &
- A bacterial platform for fermentative production of plant alkaloids. Nature Commun. 2, 326 (2011). et al.
- Genome engineering in Saccharomyces cerevisiae using CRISPR–Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).
This paper reports the use of the CRISPR–Cas system in yeast, which has the potential to markedly advance genome engineering in this organism.
- RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nature Biotech. 31, 233–239 (2013). , , , &
- Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009). et al.
- Yeast oligo-mediated genome engineering (YOGE). ACS Synth. Biol. 2, 741–749 (2013). et al.
- Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans. J. Am. Chem. Soc. 134, 4709–4720 (2012). et al.
- Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. J. Am. Chem. Soc. 134, 8212–8221 (2012). et al.
- Plasmid construction by homologous recombination in yeast. Gene 58, 201–216 (1987). , , &
- Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25, 451–452 (1997). , , &
- DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16 (2009). &
- Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster. Nature Commun. 4, 2894 (2013). et al.
- A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60-bp synthetic recombination sequences. Microb. Cell Factories 12, 47 (2013). et al.
- Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. Gene 243, 19–25 (2000). et al.
- Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE 4, e6441 (2009). &
- Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009). et al.
- Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synthet. Biol. 3, 97–106 (2014). et al.
- Automated assembly of standard biological parts. Methods Enzymol. 498, 363–397 (2011). , , &
- Synergies between synthetic biology and metabolic engineering. Nature Biotech. 29, 693–695 (2011). &
- Toward a science of metabolic engineering. Science 252, 1668–1675 (1991).
This paper provides an early description and vision of metabolic engineering.
- Metabolic engineering: past and future. Annu. Rev. Chem. Biomol. Eng. 4, 259–288 (2013). , &
- Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003). &
- DeviceEditor visual biological CAD canvas. J. Biol. Eng. 6, 1 (2012). , , , &
- j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14–21 (2012). , &
- Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools. Nucleic Acids Res. 40, e141 (2012). et al.
- PaR–PaR laboratory automation platform. ACS Synth. Biol. 2, 216–222 (2013). et al.
- Quantitative estimation of activity and quality for collections of functional genetic elements. Nature Methods 10, 347–353 (2013). et al.
- Foundations for engineering biology. Nature 438, 449–453 (2005).
- Synthetic biology: from hype to impact. Trends Biotechnol. 31, 123–125 (2013).
- Synthetic biology: new engineering rules for an emerging discipline. Mol. Syst. Biol. 2, 2006.0028 (2006). , , &
- Synthetic biology and metabolic engineering. ACS Synth. Biol. 1, 514–525 (2012).
- Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000). , &
- A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).
References 99 and 100 are two of the earliest publications on synthetic biology and demonstrate the potential to apply engineering principles to biology.
- Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995). , , , &
- Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods 10, 354–360 (2013). et al.
- Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl Acad. Sci. 109, 8884–8889 (2012). , &
- Amplifying genetic logic gates. Science 340, 599–603 (2013). , , , &
- Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol. Pharm. 5, 167–190 (2008). et al.
- 425–500 (Cold Spring Harbor Laboratory Press, 1992). , & in The Molecular and Cellular Biology of the Yeast Saccharomyces (eds Jones, E. W., Pringle, J. R. & Broach, J. R.)
- Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metab. Eng. 9, 160–168 (2006). , , , &
- Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell. Mol. Life Sci. 61, 1401–1426 (2004). , , &
- Distribution of mevalonate and glyceraldehyde 3-phosphate/pyruvate routes for isoprenoid biosynthesis in some Gram-negative bacteria and mycobacteria. FEMS Microbiol. Lett. 164, 169–175 (1998). , , &
- The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 47–65 (1999).
- Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 97, 5753–5769 (2013). et al.
- Maturation of iron–sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases. Annu. Rev. Biochem. 77, 669–700 (2008). &
- Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase. Biotechnol. Bioeng. 100, 371–378 (2008). , , &
- Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnol. Bioeng. 99, 666–677 (2008). et al.
- Roles of key active-site residues in flavocytochrome P450 BM3. Biochem. J. 339, 371–379 (1999). et al.
- Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nature Biotech. 20, 1135–1139 (2002). , &
- A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3). ACS Chem. Biol. 4, 261–267 (2009).
This paper describes the adaptation of a high-activity bacterial cytochrome P450 enzyme for the production of an oxidized artemisinin precursor.
- The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage. New Phytol. 199, 352–366 (2013). et al.