Spray-Dried Engineered Escherichia coli Whole-Cell Biocatalysts Enable Cell Recycling and In Vivo α-Ketoglutarate and NADPH Regeneration for Efficient Codeine Manufacture.

A recombinant whole-cell biocatalytic approach provides several advantages over isolated enzymes, such as improved enzyme stability, in vivo cofactor regeneration, and the facilitation of cascade reactions within a single cell. Whole-cell biocatalysis has gained attention in medicinal opioid production, due to its technical and economic advantages. In this study, recombinant Escherichia coli producing thebaine 6-O-demethylase (T6ODM), neopinone isomerase (NISO), and codeinone reductase (COR) were used to convert thebaine into codeine. Spray-drying was developed to optimize enzymatic activity in dried biocatalytic cells, achieving over 85% enzyme activity retention. These formulated cells were evaluated over multiple recycled reaction cycles that relied on in vivo cofactor regeneration. The native enzyme isocitrate dehydrogenase (IDH) regenerated both α-ketoglutarate and NADPH, critical for T6ODM and COR activity, respectively. The biocatalysts demonstrated high durability across 10 reaction cycles, with IDH and COR maintaining consistent activity. However, T6ODM exhibited a 40% reduction in activity, aligning with its lower thermal stability. The two-step bioconversion process yielded an average 68% molar yield of codeine after 10 cycles, with full thebaine consumption, demonstrating efficient cofactor and co-substrate recycling without further cellular engineering. This study highlights the potential of spray-dried E. coli cells as a sustainable and scalable approach for codeine biomanufacturing from thebaine. These results provide a strong foundation for further industrial application, offering both economic and technical benefits for large-scale opioid production.