Highlights Mutants of P450 BM3 can metabolise noscapine. Noscapine is N -demethylated with high selectivity. The metabolites produced are of interest for drug development. The profile of metabolites generated resembles that of mammalian CYP3A4.

Cytochrome P450 enzymes catalyse reactions of significant industrial interest but are underutilised in large-scale bioprocesses due to enzyme stability, cofactor requirements and the poor aqueous solubility and microbial toxicity of typical substrates and products. In this work, we investigate the potential for preparative-scale N-demethylation of the opium poppy alkaloid noscapine by a P450BM3 (CYP102A1) mutant enzyme in a whole-cell biotransformation system. We identify and address several common limitations of whole-cell P450 biotransformations using this model N-demethylation process. Mass transfer into Escherichia coli cells was found to be a major limitation of biotransformation rate and an alternative Gram-positive expression host Bacillus megaterium provided a 25-fold improvement in specific initial rate. Two methods were investigated to address poor substrate solubility. First, a biphasic biotransformation system was developed by systematic selection of potentially biocompatible solvents and in silico solubility modelling using Hansen solubility parameters. The best-performing biphasic system gave a 2.3-fold improvement in final product titre compared to a single-phase system but had slower initial rates of biotransformation due to low substrate concentration in the aqueous phase. The second strategy aimed to improve aqueous substrate solubility using cyclodextrin and hydrophilic polymers. This approach provided a fivefold improvement in initial biotransformation rate and allowed a sixfold increase in final product concentration. Enzyme stability and cell viability were identified as the next parameters requiring optimisation to improve productivity. The approaches used are also applicable to the development of other pharmaceutical P450-mediated biotransformations.

An enzymatic biosynthesis approach is described for codeine, the most widely used medicinal opiate, providing a more environmentally sustainable alternative to current chemical conversion, with yields and productivity compatible with industrial production. Escherichia coli strains were engineered to express key enzymes from poppy, including the recently discovered neopinone isomerase, producing codeine from thebaine. We show that compartmentalization of these enzymes in different cells is an effective strategy that allows active spatial and temporal control of reactions, increasing yield and volumetric productivity and reducing byproduct generation. Codeine is produced at a yield of 64% and a volumetric productivity of 0.19 g/(L·h), providing the basis for an industrially applicable aqueous whole-cell biotransformation process. This approach could be used to redirect thebaine-rich feedstocks arising from the U.S. reduction of opioid manufacturing quotas or applied to enable total biosynthesis and may have broader applicability to other medicinal plant compounds.

A whole cell Escherichia coli biotransformation platform converting thebaine to oripavine and codeine to morphine was demonstrated with industrially applicable yields (~1.2×10-2 g/(L∙h) or ~1.2×10-1 g/(L∙h)), improving > 13,400-fold upon...