Total Biosynthesis of Opiates By Engineered Escherichia coli

Opiates such as morphine, codeine, and thebaine have been used since ancient times and are the gold standard for pain relief. They are mainly obtained by extraction from opium poppies. To increase the production yields of specific alkaloids, extensive efforts have been made to characterize and subsequently modify the alkaloid biosynthetic pathway. However, the regulation of biosynthetic pathways in plants is complex; therefore, it is relatively difficult to increase production of desired products. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in the yeast system. Accordingly, microbial alkaloid production may facilitate to construct an efficient production system. The next challenge is to increase production. In this report, we demonstrate that Escherichia coli serves as an efficient, robust, and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, was produced by stepwise culture of four engineered strains at yields of 2.1 mg/L from glycerol, which corresponds to a 300-fold increase from recently developed yeast systems. This large improvement was presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we were able to biosynthesize hydrocodone, a clinically important opioid. Large improvements in opiate production in this E. coli system represent a major step toward the development of a practical opiate production system.