Article

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

California Institute of Quantitative Biomedical Research, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Nature (Impact Factor: 42.35). 05/2006; 440(7086):940-3. DOI: 10.1038/nature04640
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Jay D Keasling, Jun 18, 2015
3 Followers
 · 
273 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.
    Biotechnology Letters 05/2008; 30(4):581-92. DOI:10.1007/s10529-007-9596-y · 1.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.
    Biotechnology and Bioengineering 02/2008; 99(3):666-77. DOI:10.1002/bit.21581 · 4.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Randomness is inherent to biochemistry: at each instant, the sequence of reactions that fires is a matter of chance. Some biological systems exploit such randomness, choosing between different outcomes stochastically - in effect, hedging their bets with a portfolio of responses for different environmental conditions. In this paper, we discuss techniques for synthesizing such stochastic behavior in engineered biochemical systems. We propose a general method for designing a set of biochemical reactions that produces different combinations of molecular types according to a specified probability distribution. The response is precise and robust to perturbations. Furthermore, it is programmable: the probability distribution is a function of the quantities of input types. The method is modular and extensible. We discuss strategies for implementing various functional dependencies: linear, logarithmic, exponential, etc. This work has potential applications in domains such as biochemical sensing, drug production, and disease treatment. Moreover, it provides a framework for analyzing and characterizing the stochastic dynamics in natural biochemical systems such as the lysis/lysogeny switch of the lambda bacteriophage.
    Design Automation Conference, 2007. DAC '07. 44th ACM/IEEE; 07/2007