title: Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.
abstract: 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.
authors.affiliation: Amyris, Inc., 5885 Hollis Street, Suite 100, Emeryville, CA 94608, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Westfall
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Pitera
authors.forename: Jacob R
authors.initials: JR
authors.lastname: Lenihan
authors.forename: Diana
authors.initials: D
authors.lastname: Eng
authors.forename: Frank X
authors.initials: FX
authors.lastname: Woolard
authors.forename: Rika
authors.initials: R
authors.lastname: Regentin
authors.forename: Tizita
authors.initials: T
authors.lastname: Horning
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tsuruta
authors.forename: David J
authors.initials: DJ
authors.lastname: Melis
authors.forename: Andrew
authors.initials: A
authors.lastname: Owens
authors.forename: Scott
authors.initials: S
authors.lastname: Fickes
authors.forename: Don
authors.initials: D
authors.lastname: Diola
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Benjamin
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Michael D
authors.initials: MD
authors.lastname: Leavell
authors.forename: Derek J
authors.initials: DJ
authors.lastname: McPhee
authors.forename: Neil S
authors.initials: NS
authors.lastname: Renninger
authors.forename: Jack D
authors.initials: JD
authors.lastname: Newman
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Paddon
mesh_terms: D000962:Antimalarials / Q000737:chemistry / Q000378:metabolism*; D037621:Artemisinins / Q000737:chemistry / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D003062:Codon / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005690:Galactose / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D000081223:Polycyclic Sesquiterpenes; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D003062:Codon; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; C520462:dihydroartemisinic acid; D000431:Ethanol; C031327:artemisinin; D005947:Glucose; D005690:Galactose