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High‐level production of amorpha‐4,11‐diene in a two‐phase partitioning bioreactor of metabolically engineered Escherichia coli
Author(s) -
Newman Jack D.,
Marshall Jessica,
Chang Michelle,
Nowroozi Farnaz,
Paradise Eric,
Pitera Douglas,
Newman Karyn L.,
Keasling Jay D.
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21017
Subject(s) - bioreactor , fermentation , escherichia coli , bacteria , chemistry , industrial microbiology , biology , food science , botany , biochemistry , gene , genetics
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%. © 2006 Wiley Periodicals, Inc.