Optimized Enzyme\nProduction for the Escherichia coli Whole-Cell Biocatalytic Synthesis\nof Codeine from Thebaine

Codeine, the most widely used medicinal opiate in the\nworld, can\nbe produced from thebaine by using an environmentally friendly biocatalytic\nprocess using purified enzymes or microbial cells. However, this biotransformation\nis either inefficient or not systematically characterized for industrial\nuse, as microbial fermentation and enzyme expression have not been\noptimized, and the production of the undesirable side product neopine\nreduces yields. In this study, the expression of the required enzymes\nin a reengineeredEscherichia coli system\nwas optimized using a two-step design of experiments approach. Higher\nyields of biomass and enzyme expression were achieved in a defined\nminimal medium using lactose for induction. The interaction between\ntemperature and lactose concentration was optimized, leading to an\nimprovement in the apparent volumetric activity of thebaine 6-<i>O</i>-demethylase (T6ODM) from 45 to 776 U·L<sup>–1</sup> using glycerol as the carbon source and an improvement in the apparent\nvolumetric activity of codeinone reductase (COR) from 120 to 3707\nU·L<sup>–1</sup> using glucose. The productivity for T6ODM\nand COR was also increased 4.5 and 5.7-times to 8.0 and 28.3 U·L<sup>–1</sup>·h<sup>–1</sup>, respectively, in fed-batch\ncultivations compared to in shake flasks. With the optimized conditions\nfor enzyme expression, the whole-cell biotransformation led to an\n80% yield of codeine from thebaine and a final codeine/neopine molar\nratio of 85:15. The inclusion of coexpressed neopinone isomerase,\nwhich catalyzes the production of codeinone from neopinone, reduced\nneopine production at the start of the reaction when the concentration\nof neopinone was low but did not improve the final codeine/neopine\nratio as the reaction progressed and the concentration of neopinone\nincreased. This observation may provide a further understanding of\nthe differences between catalytic conditions in a bioreactor and the\npoppy plant, where the concentration of pathway intermediates such\nas neopinone is likely to be low and no neopine is observed. Overall,\nthe process improvements reported here provide progress toward an\nindustrial biocatalytic production of codeine from thebaine.