Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation.

[1]  G. Bàlint,et al.  Artemisinin and its derivatives: an important new class of antimalarial agents. , 2001, Pharmacology & therapeutics.

[2]  R. Hampton,et al.  Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae , 1997, Applied and environmental microbiology.

[3]  H. Michel,et al.  Heterologous expression of the human D2S dopamine receptor in protease-deficient Saccharomyces cerevisiae strains. , 1994, European journal of biochemistry.

[4]  H. Bouwmeester,et al.  Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. , 2000, Archives of biochemistry and biophysics.

[5]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[6]  R. Gardner,et al.  A Highly Conserved Signal Controls Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) Reductase in Eukaryotes* , 1999, The Journal of Biological Chemistry.

[7]  S. Picaud,et al.  Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L. , 2005, Archives of biochemistry and biophysics.

[8]  W. Quax,et al.  Seasonal Variations of Artemisinin and its Biosynthetic Precursors in Tetraploid Artemisia annua Plants Compared with the Diploid Wild-Type , 1999, Planta medica.

[9]  Nicholas J White,et al.  Antimalarial drug resistance. , 2004, The Journal of clinical investigation.

[10]  M. Brodelius,et al.  Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum. , 2002, European journal of biochemistry.

[11]  W. Quax,et al.  Isolation and identification of dihydroartemisinic acid from artemisia annua and its possible role in the biosynthesis of artemisinin , 1999, Journal of natural products.

[12]  Kevin V. Shianna,et al.  A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in Saccharomyces cerevisiae , 1998, Journal of bacteriology.

[13]  D. Pompon,et al.  Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related Arabidopsis thalianaNADPH-Cytochrome P450 Reductases with P450 CYP73A5* , 1997, The Journal of Biological Chemistry.

[14]  D. Pompon,et al.  Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae. , 1988, Gene.

[15]  X. Simonnet,et al.  The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars. , 2001, Current medicinal chemistry.

[16]  A. Antoni,et al.  A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. , 2000, Gene.

[17]  H. Bouwmeester,et al.  Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. , 2005, Planta medica.

[18]  Michael Sauer,et al.  Recombinant protein production in yeasts , 2005, Molecular biotechnology.

[19]  D. Pompon,et al.  Yeast Expression , and Characterization of the Coupling of Two Distantly Related Arabidopsis thaliana NADPH-Cytochrome P 450 Reductases with P 450 CYP 73 A 5 * , 1997 .

[20]  S. Matsuda,et al.  Metabolic engineering to produce sesquiterpenes in yeast. , 2003, Organic letters.

[21]  K. Gibson,et al.  Post-translational regulation of mevalonate kinase by intermediates of the cholesterol and nonsterol isoprene biosynthetic pathways. , 1997, Journal of lipid research.

[22]  N. Acton,et al.  A simple conversion of artemisinic acid into artemisinin. , 1989, Journal of natural products.

[23]  V. Besada,et al.  Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris , 2005, Biotechnology Letters.