A versatile yeast platform for N-demethylation of therapeutic alkaloids

Alkaloid N-demethylation is a critical step in the production of many semi-synthetic alkaloid therapeutics and has traditionally relied on synthetic chemical routes. Microbial whole-cell bioproduction offers a promising alternate route for generating these valuable pharmaceuticals. This work demonstrated a greener N-demethylation platform applicable to morphinan and tropine alkaloid classes, employing an engineered yeast expressing a cytochrome P450 enzyme with N-demethylase activity, a cytochrome P450 oxidoreductase and purine permeases as transporters. This platform enabled the N-demethylation of the morphinan alkaloids, oripavine and thebaine with high activity, and the tropane alkaloids, atropine and scopolamine with moderate activity. The study systematically evaluated the contributions of substrate uptake and enzymatic conversion within the whole-cell system. Heterologous transporters enabled mass transfer across the cell membrane, with no significant difference detected for the four substrates, while in vitro enzyme catalysis differed between substrates and correlated with yield. Computational molecular docking, combined with deep learning-based protein structure prediction, found all four substrates had high affinity for the enzyme. This approach was also used to identify potential target substrates for future biotransformation. Process performance could be further improved with the use of larger scale, well controlled bioreactors.