High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineeredEscherichia 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%.

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

“…5, compound 1) was first described from Escherichia coli which had been engineered to express the mevalonate pathway from Saccharomyces cerevisiae and amorpha-4,11-diene synthase (ADS) from A. annua (8). Development of a two-phase partitioning bioreactor allowed production of 0.5 g∕L of amorpha-4,11-diene (9). Analysis of transcriptional responses and pathway metabolites showed that 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase limited production, leading to perturbation of fatty acid biosynthesis and generalized membrane stress (10,11).…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…To reduce the loss, the secreted amorphadiene was in situ captured in dodecane organic phase in a two-phase partitioning bioreactor. [97] In contrary to the case of amorphadiene, artemisinic acid precipitates in the fermentor. Therefore, isopropyl myristate oil was supplied for solubilizing the deposited artemisinic acid.…”

Advanced biofuel production in microbes

“…Expression of MEV pathway together with and Artemisia annua amorphadiene synthase produced 122 mg/L of amorphadiene. Production of amorphadiene was increased using a two-phased partitioning system which trapped over 97% of the amorphadiene produced in the organic phase resulting in a production of 480 mg/L of amorphadiene [31].…”