Synthetic biology

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

Journal name:
Nature Reviews Microbiology
Volume:
12,
Pages:
355–367
Year published:
DOI:
doi:10.1038/nrmicro3240
Published online

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.

At a glance

Figures

  1. Artemisinin biosynthesis pathway in the plant Artemisia annua.
    Figure 1: Artemisinin biosynthesis pathway in the plant Artemisia annua.

    Sugars that are produced by photosynthesis in plant chloroplasts are converted to acetyl-CoA in the cytosol. The two-carbon acetyl-CoA feeds into the cytosolic mevalonate pathway, which produces farnesyl diphosphate (FPP). FPP is converted to the 15-carbon sesquiterpene amorphadiene by the enzyme amorphadiene synthase (ADS). Amorphadiene is subsequently enzymatically oxidized to either artemisinic acid or dihydroartemisinic acid; different chemotypes of A. annua produce different ratios of these products30, 118. Dihydroartemisinic acid is the precursor of artemisinin and is thought to be converted to artemisinin in a non-enzymatic photochemical reaction that is stimulated by sunlight28.

  2. The main stages involved in the synthesis of semi-synthetic artemisinin.
    Figure 2: The main stages involved in the synthesis of semi-synthetic artemisinin.

    The initial stage involved the engineering of the parent Saccharomyces cerevisiae strain to produce >25 g per L amorphadiene by overexpression of nine genes of the mevalonate pathway and expression of the Artemisia annua amorphadiene synthase. Subsequent engineering steps, involving expression of the A. annua cytochrome P450 enzyme, CYP71AV1, its cognate reductase CPR1, cytochrome b5 and two dehydrogenases enabled the oxidation of amorphadiene to artemisinic acid, which was extracted from fermentation broth and chemically converted to artemisinin. The maximum titre achieved using this procedure was 25 g per L artemisinic acid. Figure is modified, with permission, from Ref. 55 © Macmillan Publisters Ltd. All rights reserved.

  3. Engineering approaches required for pathway construction and semi-synthetic artemisinin production.
    Figure 3: Engineering approaches required for pathway construction and semi-synthetic artemisinin production.

    Escherichia coli was initially used as the chassis organism for the optimization of the mevalonate expression pathway, but steps subsequent to amorphadiene synthesis were difficult to recapitulate in E. coli, and Saccharomyces cerevisiae was therefore used as the chassis organism for engineering the entire synthesis pathway. The mevalonate pathway was heterologously expressed in E. coli on two plasmids (MevB and MevT), along with a plasmid encoding the Artemisia annua amorphadiene synthase (ADS), which enabled the conversion of acetyl-CoA to amorphadiene. The yeast strain was engineered to overexpress the mevalonate pathway, and all genes were integrated into the genome, with the exception of ADS and CYP71AV1, which were plasmid-borne, and were required for the conversion of amorphadeine to artemisinic alcohol. Chromosomal insertion of two more genes (ADH1 and ALDH1) was required for conversion to artemisinic acid. The final step of the pathway, which is the conversion of artemisinic acid to artemisinin, required chemical conversion. The genes expressed encode the following enzymes: ADH1, artemisinic alcohol dehydrogenase; ADS, amorphadiene synthase; ALDH1, artemisinic aldehyde dehydrogenase; atoB and ERG10, Acetoacetyl-CoA thiolase; CPR1, cytochrome P450 reductase; CYB5, cytochrome b5; CYP71AV1, cytochrome P450 enzyme that converts amorphadiene to artemisinic alcohol; ERG8, phosphomevalonate kinase; ERG12, mevalonate kinase; ERG13 and mvaS, HMG-CoA synthase; idi and IDI1, isopentenyl diphosphate isomerase; ispA and ERG20, farnesy diphosphate (FPP) synthase; mvaA, HMG-CoA reductase; MVD1, mevalonate diphosphate decarboxylase; tHMG1, truncated HMG-CoA reductase (tHMGR). Genes coloured blue are derived from E. coli, yellow genes are derived from S. cerevisiae, purple genes are derived from Staphylococcus aureus and green genes are derived from A. annua. All A. annua genes were synthetic and codon-optimized for the chassis organism.

  4. Molecules amenable to synthetic production using similar principles to those described for semi-synthetic artemisinin.
    Figure 4: Molecules amenable to synthetic production using similar principles to those described for semi-synthetic artemisinin.

    These molecules include vincristine, a vinca alkaloid; morphine, a benzyl isoquinoline alkaloid; prostratin, an isoprenoid; paclitaxel, an isoprenoid and bleomycin, a glycopeptide. Vincristine and paclitaxel are anticancer drugs, morphine is an analgesic, prostratin has anti-HIV properties and bleomycin is an antibiotic.

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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

Competing interests statement

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

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Author details

  • Chris J. Paddon

    Chris J. Paddon is a principal scientist at Amyris, Inc., California, USA, and was project leader for the Semi-synthetic Artemisinin Project. After graduating from the University of Surrey, he obtained a Ph.D. from Imperial College, London, UK. Following postdoctoral work at the US National Institutes of Health (NIH), Bethesda, Maryland, USA, he worked for several pharmaceutical and biopharmaceutical companies before joining Amyris, Inc. — a leading renewable fuels and chemicals company and a pioneer of industrial synthetic biology.

  • Jay D. Keasling

    Jay D. Keasling is the Hubbard Howe Jr. Distinguished Professor of Biochemical Engineering at the University of California, Berkeley, USA, in the Department of Bioengineering and Chemical and Biomolecular Engineering, Senior Faculty Scientist and Associate Laboratory Director for Biosciences at Lawrence Berkeley National Laboratory, Berkeley, California, USA, Chief Executive Officer of the Joint BioEnergy Institute (JBEI), Emeryville, California, USA, and director of the Synthetic Biology Engineering Research Center (SynBERC), Berkeley, California, USA. His research focuses on the metabolic engineering of microorganisms for the degradation of environmental contaminants and for the environmentally friendly synthesis of drugs, chemicals and fuels.

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