dihydroartemisinic-acid and amorpha-4-11-diene

Topics: Aldehyde Dehydrogenase 1 Family; Artemisinins; Biocatalysis; Biosynthetic Pathways; Cytochrome P-450 Enzyme System; Fungal Proteins; Isoenzymes; Oxidation-Reduction; Polycyclic Sesquiterpenes; Retinal Dehydrogenase; Saccharomyces cerevisiae; Sesquiterpenes

Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using

Topics: Antimalarials; Artemisia annua; Artemisinins; Biosynthetic Pathways; Crosses, Genetic; DNA, Plant; Gene Dosage; Genotype; Mutagenesis; Mutation; Plant Leaves; Plant Proteins; Polycyclic Sesquiterpenes; Polymorphism, Single Nucleotide; Sesquiterpenes; Terpenes; Trichomes

Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

Topics: Antimalarials; Artemisinins; Batch Cell Culture Techniques; Codon; Ethanol; Fermentation; Galactose; Genes, Fungal; Genotype; Glucose; Polycyclic Sesquiterpenes; Saccharomyces cerevisiae; Sesquiterpenes

Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.

Topics: Algorithms; Artemisinins; Bacillus megaterium; Bacterial Proteins; Catalytic Domain; Computer Simulation; Crystallography, X-Ray; Cytochrome P-450 Enzyme System; Models, Molecular; Molecular Conformation; Mutation; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Polycyclic Sesquiterpenes; Protein Engineering; Sesquiterpenes; Stereoisomerism; Time Factors