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.

Metastatic tumors in the brain represent one of the leading causes of death from cancer with current treatments being largely ineffective and/or associated with significant side effects due to their lack of targeting. Conjugating MRI contrast agents with a monoclonal antibody for VCAM1 (anti-VCAM1) has previously allowed detection of brain tumor volumes two to three orders of magnitude smaller than those volumes currently detectable clinically. In this study, a novel magnetic and acoustically responsive phospholipid-stabilised nanodroplet formulation has been conjugated with anti-VCAM-1. Preliminary in vivo tests have shown that these anti-VCAM-1 nanodroplets can be successfully targeted to both inflamed areas of the brain and metastatic brain tumours. Acoustic droplet vaporisation of the anti-VCAM1 droplets, confirmed by passive cavitation detection, was also shown to cause blood brain barrier permeabilisation in vivo. Extensive in vitro characterisation of the potential of these nanodroplets to target brain metastases using antibody and magnetic targeting, along with the ultrasound conditions required for various therapeutic effects has been completed and has been found to agree well with mathematical modelling. The implications of these findings and the plans for future investigations into the therapeutic potential of these targeted and ultrasound responsive agents will be discussed.