Codeine 3-O-demethylase catalyzed biotransformation of morphinan alkaloids in Escherichia coli: site directed mutagenesis of terminal residues improves enzyme expression, stability and biotransformation yield.

The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morphinan alkaloids by O-demethylation and has been applied in single step biotransformation reactions or as part of larger biosynthetic cascade, however, the productivity for these reactions remains low and suboptimal enzyme properties could be improved. This mutagenesis study targeted non-conserved N-and C-terminal residues, which were replaced with the equivalent residues from enzyme thebaine 6-O-demethylase. Whole cell biotransformation performance was significantly improved in Escherichia coli expressing codeine 3-O-demethylase mutants, with a ~ 2.8-fold increase in the production of oripavine from thebaine and ~ 1.3-fold increase in the production of morphine from codeine. Statistical analysis of biotransformation yield, enzyme expression and stability, predicted using changes in Gibbs free energy (ΔΔG) with deep-learning-based model DDmut, suggested that altered enzyme stability and/or expression of soluble protein may contribute to the observed improvements in biotransformation. This approach could be beneficial for screening future codeine 3-O-demethylase mutations and for other enzymes.