High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli

✍ Scribed by Jack D. Newman; Jessica Marshall; Michelle Chang; Farnaz Nowroozi; Eric Paradise; Douglas Pitera; Karyn L. Newman; Jay D. Keasling

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 168 KB
• Volume: 95
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.21017

✦ Synopsis

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, ninegene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the antimalarial 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%.