Review Article | Published:

Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development

Nature Reviews Microbiology volume 12, pages 355367 (2014) | Download Citation

Abstract

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.

Key points

  • Artemisinin is an antimalarial drug precursor that is produced by the plant Artemisia annua. The supply and price of artemisinin have fluctuated substantially throughout the past decade, owing to inconsistencies in harvest.

  • Artemisinin-based combination therapies (ACTs) are recommended by the WHO as the first-line treatment for uncomplicated malaria. The Semi-synthetic Artemisinin Project aimed to stabilize the supply and price of artemisinin for the development of artemisinin derivatives for use as part of ACTs.

  • Both Escherichia coli and Saccharomyces cerevisiae were engineered using the tools of synthetic biology to produce 25 g per L and 40 g per L, respectively, of the artemisinin hydrocarbon precursor amorphadiene by fermentation.

  • Owing to problems using E. coli, S. cerevisiae was used as the chassis for the industrial-scale production of 25 g per L artemisinic acid by fermentation, which was followed by a chemical conversion process to synthesize artemisinin.

  • Semi-synthetic artemisinin is now produced at industrial scale and has been approved by the WHO for the preparation of approved pharmaceutical compounds for incorporation into ACTs.

  • Lessons learned from the Semi-synthetic Artemisinin Project that are relevant to the development of other pharmaceutical products using metabolic engineering and synthetic biology are summarized.

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References

  1. 1.

    Aspirin and Related Drugs (CRC Press, 2004).

  2. 2.

    Working Knowledge: aspirin. Sci. Am. 280, 108 (1999).

  3. 3.

    Untersuchungen über die wasserfreien organischen Säuren. Justus Liebigs Annalen Chemie 87, 57–84 (in German) (1853).

  4. 4.

    Qinghaosu (artemisinin): the price of success. Science 320, 330–334 (2008). A review of the history and properties of artemisinin.

  5. 5.

    & How Chinese scientists discovered qinghaosu (artemisinin) and developed its derivatives? What are the future perspectives? Med. Trop. 58, 9–12 (1998).

  6. 6.

    World Health Organisation. Guidelines for the treatment of malaria. 2nd edn (WHO, 2010).

  7. 7.

    World Health Organisation. WHO informal consultation with manufacturers of artemisinin-based pharmaceutical products in use for the treatment of malaria (WHO, 2007).

  8. 8.

    , , & 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).

  9. 9.

    World Health Organisation. World Malaria Report 2012 (WHO, 2012).

  10. 10.

    Reflections on the 'discovery' of the antimalarial qinghao. Br. J. Clin. Pharmacol. 61, 666–670 (2006).

  11. 11.

    Qinghaosu Antimalarial Coordinating Research Group. Antimalaria Studies on Qinghaosu. Chin. Med. J. 12, 811–816 (1979).

  12. 12.

    Qinghaosu (artemisinin): an antimalarial drug from China. Science 228, 1049–1055 (1985).

  13. 13.

    Artemisia Annua, artemisinin, ACTs & malaria control in Africa. Tradition, science and public policy , (2012).

  14. 14.

    & Discovery, mechanisms of action and combination therapy of artemisinin. Expert Rev. Anti-Infective Ther. 7, 999–1013 (2009).

  15. 15.

    , & 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).

  16. 16.

    et al. A major genome region underlying artemisinin resistance in Malaria. Science 336, 79–82 (2012).

  17. 17.

    et al. Artemisinin resistance: current status and scenarios for containment. Nature Rev. Microbiol. 8, 272–280 (2010).

  18. 18.

    et al. Artemisinin-resistant Malaria: research challenges, opportunities, and public health implications. Am. J. Trop. Med. Hyg. 87, 231–241 (2012).

  19. 19.

    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).

  20. 20.

    et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nature Genet. 45, 648–655 (2013).

  21. 21.

    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.

  22. 22.

    & Stabilizing supply of artemisinin and artemisinin-based combination therapy in an era of wide-spread scale-up. Malar J. 11, 399 (2012).

  23. 23.

    Dahlberg Global Development Advisors. Independent Mid-Term review of the assured Artemisinin Supply System (A2S2) Project. Geneva: UNITAID , (2012).

  24. 24.

    Assured Artemisinin Supply System. Artemisinin imports into India , (updated 13 January 2014).

  25. 25.

    WHO Health Financing. Per capita total expenditure on health at average exchange rate (US $) , (2011).

  26. 26.

    The World Bank. Health expenditure per capita (current US$). (2013).

  27. 27.

    et al. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–47 (2005).

  28. 28.

    The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao). Molecules 15, 7603–7698 (2010).

  29. 29.

    et al. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 52, 843–854 (1999).

  30. 30.

    , , , & Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68, 1864–1871 (2007).

  31. 31.

    et al. In Isoprenoid Synthesis in Plants and Microorganisms (eds Bach, T. J. & Rohmer, M.) 91–106 (Springer, 2013).

  32. 32.

    et al. 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).

  33. 33.

    , , , & Methylerythritol phosphate pathway of isoprenoid biosynthesis. Annu. Rev. Biochem. 82, 497–530 (2013).

  34. 34.

    et al. Mono and diterpene production in Escherichia coli. Biotechnol. Bioeng. 87, 200–212 (2004).

  35. 35.

    , , , & 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.

  36. 36.

    , & The in vivo synthesis of plant sesquiterpenes by Escherichia coli. Biotechnol. Bioeng. 75, 497–503 (2001).

  37. 37.

    et al. 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).

  38. 38.

    , , & Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9, 193–207 (2007).

  39. 39.

    , , & Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nature Biotech. 24, 1027–1032 (2006).

  40. 40.

    , , , & Microbial sensors for small molecules: development of a mevalonate biosensor. Metab. Eng. 9, 30–38 (2007).

  41. 41.

    , , & Application of functional genomics to pathway optimization for increased isoprenoid production. Appl. Environ. Microbiol. 74, 3229–3241 (2008).

  42. 42.

    & Production of mevalonate by a metabolically-engineered Escherichia coli. Biotechnol. Lett. 26, 1487–1491 (2004).

  43. 43.

    , , & Class II 3-hydroxy-3-methylglutaryl coenzyme A reductases. J. Bacteriol. 186, 1927–1932 (2004).

  44. 44.

    et al. 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.

  45. 45.

    & A simple conversion of artemisinic acid into artemisinin. J. Nature Prod. 52, 1183–1185 (1989).

  46. 46.

    & Simple conversion of artemisinic acid into artemisinin. US Patent 4992561 (1991).

  47. 47.

    et al. 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.

  48. 48.

    , , , & Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nature Chem. Biol. 3, 274–277 (2007).

  49. 49.

    , , , & 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).

  50. 50.

    & Genealogy of principal strains of the yeast genetic stock center. Genetics 113, 35–43 (1986).

  51. 51.

    et al. Life with 6000 genes. Science 274, 563–567 (1996).

  52. 52.

    et al. Four linked genes participate in controlling sporulation efficiency in budding yeast. PLoS Genet. 2, e195 (2006).

  53. 53.

    et al. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26, 706–714 (2000).

  54. 54.

    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.

  55. 55.

    et al. 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.

  56. 56.

    et al. 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).

  57. 57.

    , & Mechanisms that regulate production of reactive oxygen species by cytochrome P450. Toxicol. Appl. Pharmacol. 199, 316–331 (2004).

  58. 58.

    , , , & 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).

  59. 59.

    & The many roles of cytochrome b5. Pharmacol. Ther. 97, 139–152 (2003).

  60. 60.

    , & 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).

  61. 61.

    , , & 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.

  62. 62.

    Sanofi. Prix Potier 2012 (in French) , (2012).

  63. 63.

    WHO Prequalificaion of Medicines Programme. Acceptance of non-plant-derived-artemisinin offers potential to increase access to malaria treatment , (2013).

  64. 64.

    et al. Isoprenoid pathway optimization for taxol precursor overproduction in E. coli. Science 330, 70–74 (2010).

  65. 65.

    , & Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab. Eng. 10, 201–206 (2008).

  66. 66.

    & Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nature Chem. Biol. 4, 564–573 (2008).

  67. 67.

    et al. Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl Acad. Sci. USA 105, 7393–7398 (2008).

  68. 68.

    , & Recent progress in the metabolic engineering of alkaloids in plant systems. Curr. Opin. Biotechnol. 24, 354–365 (2013).

  69. 69.

    et al. A bacterial platform for fermentative production of plant alkaloids. Nature Commun. 2, 326 (2011).

  70. 70.

    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.

  71. 71.

    , , , & RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nature Biotech. 31, 233–239 (2013).

  72. 72.

    et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

  73. 73.

    et al. Yeast oligo-mediated genome engineering (YOGE). ACS Synth. Biol. 2, 741–749 (2013).

  74. 74.

    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).

  75. 75.

    et al. Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. J. Am. Chem. Soc. 134, 8212–8221 (2012).

  76. 76.

    , , & Plasmid construction by homologous recombination in yeast. Gene 58, 201–216 (1987).

  77. 77.

    , , & Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25, 451–452 (1997).

  78. 78.

    & DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16 (2009).

  79. 79.

    et al. Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster. Nature Commun. 4, 2894 (2013).

  80. 80.

    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).

  81. 81.

    et al. Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. Gene 243, 19–25 (2000).

  82. 82.

    & Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE 4, e6441 (2009).

  83. 83.

    et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009).

  84. 84.

    et al. Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synthet. Biol. 3, 97–106 (2014).

  85. 85.

    , , & Automated assembly of standard biological parts. Methods Enzymol. 498, 363–397 (2011).

  86. 86.

    & Synergies between synthetic biology and metabolic engineering. Nature Biotech. 29, 693–695 (2011).

  87. 87.

    Toward a science of metabolic engineering. Science 252, 1668–1675 (1991). This paper provides an early description and vision of metabolic engineering.

  88. 88.

    , & Metabolic engineering: past and future. Annu. Rev. Chem. Biomol. Eng. 4, 259–288 (2013).

  89. 89.

    & Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003).

  90. 90.

    , , , & DeviceEditor visual biological CAD canvas. J. Biol. Eng. 6, 1 (2012).

  91. 91.

    , & j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14–21 (2012).

  92. 92.

    et al. Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools. Nucleic Acids Res. 40, e141 (2012).

  93. 93.

    et al. PaR–PaR laboratory automation platform. ACS Synth. Biol. 2, 216–222 (2013).

  94. 94.

    et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nature Methods 10, 347–353 (2013).

  95. 95.

    Foundations for engineering biology. Nature 438, 449–453 (2005).

  96. 96.

    Synthetic biology: from hype to impact. Trends Biotechnol. 31, 123–125 (2013).

  97. 97.

    , , & Synthetic biology: new engineering rules for an emerging discipline. Mol. Syst. Biol. 2, 2006.0028 (2006).

  98. 98.

    Synthetic biology and metabolic engineering. ACS Synth. Biol. 1, 514–525 (2012).

  99. 99.

    , & Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

  100. 100.

    & 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.

  101. 101.

    , , , & Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).

  102. 102.

    et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods 10, 354–360 (2013).

  103. 103.

    , & Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl Acad. Sci. 109, 8884–8889 (2012).

  104. 104.

    , , , & Amplifying genetic logic gates. Science 340, 599–603 (2013).

  105. 105.

    et al. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol. Pharm. 5, 167–190 (2008).

  106. 106.

    , & in The Molecular and Cellular Biology of the Yeast Saccharomyces (eds Jones, E. W., Pringle, J. R. & Broach, J. R.) 425–500 (Cold Spring Harbor Laboratory Press, 1992).

  107. 107.

    , , , & Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metab. Eng. 9, 160–168 (2006).

  108. 108.

    , , & Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell. Mol. Life Sci. 61, 1401–1426 (2004).

  109. 109.

    , , & 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).

  110. 110.

    The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 47–65 (1999).

  111. 111.

    et al. Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 97, 5753–5769 (2013).

  112. 112.

    & Maturation of iron–sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases. Annu. Rev. Biochem. 77, 669–700 (2008).

  113. 113.

    , , & Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase. Biotechnol. Bioeng. 100, 371–378 (2008).

  114. 114.

    et al. Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnol. Bioeng. 99, 666–677 (2008).

  115. 115.

    et al. Roles of key active-site residues in flavocytochrome P450 BM3. Biochem. J. 339, 371–379 (1999).

  116. 116.

    , & Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nature Biotech. 20, 1135–1139 (2002).

  117. 117.

    et al. 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.

  118. 118.

    et al. 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).

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Acknowledgements

The authors thank the Bill and Melinda Gates Foundation for their generous support of this project. They also thank their many colleagues at the University of California Berkeley, Amyris, Sanofi, National Research Council of Canada (NRC) and PATH Drug Solutions for their unstinting efforts that made this work a success.

Author information

Affiliations

  1. Amyris, Inc., 5885 Hollis Street, Suite 100, Emeryville, California 94608, USA.

    • Chris J. Paddon
  2. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, USA.

    • Jay D. Keasling
  3. Department of Bioengineering, University of California, Berkeley, California 94720, USA.

    • Jay D. Keasling
  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

    • Jay D. Keasling
  5. Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, California 94608, USA.

    • Jay D. Keasling

Authors

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Competing interests

C.J.P. and J.D.K. hold stock options and shares in Amyris Inc. (California, USA).

Corresponding authors

Correspondence to Chris J. Paddon or Jay D. Keasling.

Glossary

Chassis organism

The host microorganism that is used for the production of a desired product; it is typically subjected to genetic manipulation using the tools of synthetic biology.

Tunable intergenic regions

(TIGRs). Stretches of DNA that are located between genes and that can be modified (for example, by the insertion of hairpins and cleavage sites) to enable each gene in an operon to be varied independently of all others in a random manner.

Fed-batch fermentation process

A batch culture that is fed continuously with nutrient medium. It differs from continuous culture owing to the variation in culture volume from beginning to end.

Cytochrome P450 enzyme

A member of a superfamily of monooxygenase enzymes that catalyse the oxidation of organic substrates. Plant microsomal cytochrome P450 enzymes require a reductase enzyme (CPR) to transfer electrons from NADPH and may also require cytochrome b5 to supply electrons.

Trichomes

Hair-like or glandular structures on the surface of plants. Glandular trichomes are the major biosynthetic site of many natural plant products, including artemisinin.

Hock fragmentation

A reaction of hydroperoxides connected to an unsaturated system, leading to cleavage of the C–C bond and the formation of two carbonyl compounds.

Photochemical conversion

A chemical reaction that is initiated by the absorption of energy in the form of visible, ultraviolet or infrared light.

Alkaloids

Nitrogen-containing natural products, many of which have pharmacological properties. Examples include cocaine, caffeine and the antimalarial drug quinine.

Chemotypes

Plants that are morphologically similar or identical but that are distinguished by differences in the production of secondary metabolites.

Yeast homologous recombination

A method to assemble DNA fragments with overlapping homology regions by transforming yeast and using its innate homologous recombination ability to correctly assemble the fragments.

Ligase chain reaction

(LCR). A method to assemble DNA fragments using bridging oligonucleotides, a thermostable DNA polymerase and multiple denaturation–annealing–ligation temperature cycles.

Circular polymerase extension cloning

(CPEC). A polymerase chain reaction (PCR)-based method for the assembly of multiple DNA fragments.

Gibson isothermal assembly

A single-temperature enzymatic method of assembling multiple DNA fragments.

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