ABSTRACT
In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.
Subject(s)
Artemisinins/metabolism , Artemisinins/supply & distribution , Biosynthetic Pathways , Saccharomyces cerevisiae/metabolism , Antimalarials/economics , Antimalarials/isolation & purification , Antimalarials/metabolism , Antimalarials/supply & distribution , Artemisinins/chemistry , Artemisinins/economics , Artemisinins/isolation & purification , Biotechnology , Fermentation , Genetic Engineering , Malaria, Falciparum/drug therapy , Molecular Sequence Data , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Singlet Oxygen/metabolismABSTRACT
Members of the septin gene family are involved in cytokinesis and the organization of new growth in organisms as diverse as yeast, fruit fly, worm, mouse, and human. Five septin genes have been cloned and sequenced from the model filamentous fungus A. nidulans. As expected, the A. nidulans septins contain the highly conserved GTP binding and coiled-coil domains seen in other septins. On the basis of hybridization of clones to a chromosome-specific library and correlation with an A. nidulans physical map, the septins are not clustered but are scattered throughout the genome. In phylogenetic analysis most fungal septins could be grouped with one of the prototypical S. cerevisiae septins, Cdc3, Cdc10, Cdc11, and Cdc12. Intron-exon structure was conserved within septin classes. The results of this study suggest that most fungal septins belong to one of four orthologous classes.
Subject(s)
Aspergillus nidulans/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Aspergillus nidulans/growth & development , Blotting, Northern , Cloning, Molecular , Cytoskeletal Proteins/physiology , DNA, Complementary/metabolism , Databases, Factual , Exons , Guanosine Triphosphate/metabolism , Introns , Models, Biological , Models, Genetic , Molecular Sequence Data , Multigene Family , Mutation , Nucleic Acid Hybridization , Phylogeny , Physical Chromosome Mapping , Profilins , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Time FactorsABSTRACT
When the spores of filamentous fungi break dormancy, they grow isotropically, adding cell wall material uniformly in every direction. Later they switch to polarized growth, with new material added to the tip of an emerging germ tube. To identify genes involved in the synthesis and localization of cell wall material in filamentous fungi, we screened a collection of temperature-sensitive Aspergillus nidulans mutants for swollen cells. We have isolated mutants representing eight genes involved in polarity establishment, polarity maintenance, and hyphal morphogenesis. On the basis of the results of temperature-shift experiments, swo C, D, and F are required to establish polarity, while swoA is required to maintain polarity. swo B, E, G, and H are involved in later hyphal morphogenesis. Our results suggest that polarity establishment and polarity maintenance are genetically separate events and that a persistent signal is required for apical extension in A. nidulans.
