Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM et al.. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940-943

California Institute of Quantitative Biomedical Research, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Nature (Impact Factor: 41.46). 05/2006; 440(7086):940-3. DOI: 10.1038/nature04640
Source: PubMed


Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

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    • "Recent proof-of-concept applications of synthetic biology toward the partial reconstruction of plant secondary metabolic pathways in Escherichia coli and yeast (Saccharomyces cerevisiae) has underscored the potential of using microbes as alternative platforms (Ro et al., 2006; Minami et al., 2008; Hawkins and Smolke, 2008; Engels et al., 2008; Ajikumar et al., 2010; Fossati et al., 2014; Thodey et al., 2014). Among these, several noscapine pathway intermediates including (S)-reticuline, (S)-scoulerine, (S)-tetrahydrocolumbamine and (S)-canadine have been produced using different strategies (Minami et al., 2008; Hawkins and Smolke, 2008; Nakagawa et al., 2011). "
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    ABSTRACT: Noscapine is a phthalideisoquinoline alkaloid, which represents a class of plant specialized metabolites within the large and structurally diverse group of benzylisoquinoline alkaloids. Along with the narcotic analgesic morphine, noscapine is a major alkaloid in the latex of opium poppy (Papaver somniferum) that has long been used as a cough suppressant and has undergone extensive investigation as a potential anticancer drug. Cultivated opium poppy plants remain the only commercial source of noscapine. Despite its isolation from opium more than two centuries ago, the almost complete biosynthesis of noscapine has only recently been established based on an impressive combination of molecular genetics, functional genomics, and metabolic biochemistry. In this review, we provide a historical account of noscapine from its discovery through to initial investigations of its formation in opium poppy. We also describe recent breakthroughs that have led to an elucidation of the noscapine biosynthetic pathway, and we discuss the pharmacological properties that have prompted intensive evaluation of the potential pharmaceutical applications of noscapine and several semi-synthetic derivatives. Finally, we speculate on the future potential for the production of noscapine using metabolic engineering and synthetic biology in plants and microbes.
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    • "Although great progress has been made in dissecting artemisinin biosynthesis and reconstruction of artemisinin biosynthesis in microorganisms (Ro et al., 2006; Dietrich et al., 2009), the regulatory mechanisms of artemisinin biosynthesis in planta are still poorly understood. AaWRKY1, a WRKY transcription factor, was reported to function as a positive regulator of artemisinin biosynthesis by binding to the W-box in the ADS promoter (Ma et al., 2009a); AaERF1 and AaERF2, belonging to the AP2/ERF transcription factors family, positively regulate the transcription of ADS and CYP71AV1 by binding to the RAA and CBF2 motif (Yu et al., 2012); AaORA, a trichome-specific AP2/ERF transcription factor, enhances biosynthesis of artemisinin when overexpressed (Lu et al., 2013b). "
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    ABSTRACT: Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production.
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    • "with permission from Nature (Ro et al. 2006) ©(2006) Macmillan Publishers Ltd "
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    ABSTRACT: Number of microorganisms produces antibiotics that can inhibit or kill the other microbes. The production of some antibiotics is not sufficient in native host rather difficult to synthesize chemically and to extract in large amounts for commercialization. Metabolic engineering plays an increasingly significant role in the production of antibiotics and its precursors. Thus, we engineer biosynthetic pathways in desire host for the production of sufficient quantity of antibiotics. In this chapter, we illustrated bioengineering of different microbes using synthetic biology and metabolic engineering approaches for production and regulation of antibiotics.
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