Yeast based N-demethylation for the production of nororipavine in fed-batch or continuous cultivation

• An insect N -demethylase was expressed in Saccharomyces cerevisiae. • Whole cell bioconversion of oripavine to nororipavine was achieved. • Fed-batch and continuous fermentation effectively produce nororipavine. • A high titre (1.59 g/L) and time–space-yield (1.01 g/L/day) was obtained. • This process could replace the N -demethylation step used to make buprenorphine. Buprenorphine can assist in the treatment of opiate addiction but the current methods of synthesis for this alkaloid are suboptimal. N -demethylation of the precursor oripavine by bioconversion could present an alternative route for the first steps of synthesis but requires an N -demethylase that can efficiently produce nororipavine at sufficient yields, together with a process that can be controlled and scaled within a bioreactor. An N -demethylase acting on oripavine was sourced from insect pest species living on the alkaloid-containing parts of poppy that naturally contain oripavine. When coupled with a cytochrome P450 oxidoreductase and purine permease for transport, the enzyme expressed in Saccharomyces cerevisiae could N -demethylate oripavine to nororipavine. Bioconversion examined in batch/fed-batch mode gave a greater understanding of the optimal concentration of oripavine substrate and kinetics of nororipavine production. The highest titre of 1.59 g/L was achieved with fed-batch cell growth and batch bioconversion, due to greater cell biomass and improved process control, while continuous processing gave a productivity of 1.01 g/L/day at 0.7 L scale. This study presents an alternative approach to achieving an environmentally friendly, industrially scalable whole-cell conversion of high-value benzylisoquinoline alkaloids using a novel N -demethylase. Yield and productivity are promising, with potential for further optimisation of both enzyme and bioconversion process, including further exploration of a continuous process for process intensification. The N -demethylation step described here could prove useful in the production of buprenorphine or be coupled with other described whole cell pathways for more complex conversions of therapeutic alkaloids.