Optimized Enzyme Production for the Escherichia coli Whole-Cell Biocatalytic Synthesis of Codeine from Thebaine

Codeine, the most widely used medicinal opiate in the world, can be produced from thebaine by using an environmentally friendly biocatalytic process using purified enzymes or microbial cells. However, this biotransformation is either inefficient or not systematically characterized for industrial use, as microbial fermentation and enzyme expression have not been optimized, and the production of the undesirable side product neopine reduces yields. In this study, the expression of the required enzymes in a reengineeredEscherichia coli system was optimized using a two-step design of experiments approach. Higher yields of biomass and enzyme expression were achieved in a defined minimal medium using lactose for induction. The interaction between temperature and lactose concentration was optimized, leading to an improvement in the apparent volumetric activity of thebaine 6-O-demethylase (T6ODM) from 45 to 776 U·L^–1 using glycerol as the carbon source and an improvement in the apparent volumetric activity of codeinone reductase (COR) from 120 to 3707 U·L^–1 using glucose. The productivity for T6ODM and COR was also increased 4.5 and 5.7-times to 8.0 and 28.3 U·L^–1·h^–1, respectively, in fed-batch cultivations compared to in shake flasks. With the optimized conditions for enzyme expression, the whole-cell biotransformation led to an 80% yield of codeine from thebaine and a final codeine/neopine molar ratio of 85:15. The inclusion of coexpressed neopinone isomerase, which catalyzes the production of codeinone from neopinone, reduced neopine production at the start of the reaction when the concentration of neopinone was low but did not improve the final codeine/neopine ratio as the reaction progressed and the concentration of neopinone increased. This observation may provide a further understanding of the differences between catalytic conditions in a bioreactor and the poppy plant, where the concentration of pathway intermediates such as neopinone is likely to be low and no neopine is observed. Overall, the process improvements reported here provide progress toward an industrial biocatalytic production of codeine from thebaine.