title: High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli.
abstract: Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.
authors.affiliation: Department of Chemical Engineering, University of California, Berkeley, California, USA.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Newman
authors.forename: Jessica
authors.initials: J
authors.lastname: Marshall
authors.forename: Michelle
authors.initials: M
authors.lastname: Chang
authors.forename: Farnaz
authors.initials: F
authors.lastname: Nowroozi
authors.forename: Eric
authors.initials: E
authors.lastname: Paradise
authors.forename: Douglas
authors.initials: D
authors.lastname: Pitera
authors.forename: Karyn L
authors.initials: KL
authors.lastname: Newman
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000962:Antimalarials / Q000138:chemical synthesis; D037621:Artemisinins / Q000378:metabolism*; D019149:Bioreactors; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology*; D005740:Gases / Q000302:isolation & purification*; D006207:Half-Life; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000302:isolation & purification / Q000378:metabolism; D013729:Terpenes / Q000302:isolation & purification / Q000378:metabolism*; D014835:Volatilization
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D005740:Gases; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; D013729:Terpenes; C515348:amorpha-4,11-diene