Biotechnological synthesis of drug metabolites using human cytochrome P450 2D6 heterologously expressed in fission yeast exemplified for the designer drug metabolite 4′-hydroxymethyl-α-pyrrolidinobutyrophenone

The aim of this study was evaluating the principle feasibility of biotechnological synthesis of drug metabolites using heterologously expressed human cytochrome P450 (CYP) enzymes. Human CYP2D6 expressed in fission yeast (Schizosaccharomyces pombe) strain CAD58 was used as model enzyme and the designer drug 4′-methyl-α-pyrrolidinobutyrophenone (MPBP) as model drug. For synthesis of 4′-hydroxmethyl-α-pyrrolidinobutyrophenone (HO-MPBP), 250 μmol of MPBP·HNO3 were incubated with one litre of CAD58 culture (108 cells/mL, pH 9, 48 h, 30 °C). HO-MPBP was isolated by liquid-liquid extraction and precipitated as its hydrochloride salt. Identity and purity of the product were tested by HPLC with ultraviolet (UV) detection, GC-MS, and 1H-NMR. CAD58 was further characterized regarding the influence of incubation pH (5-10), cell density (107-108 cells/mL), and incubation time (0-120 h) on metabolite formation using the substrates dextromethorphan and MPBP. The preparative experiment yielded 40 mg (141 μmol) of HO-MPBP·HCl with a purity of >98%. In the characterization experiments, the metabolite formation rate peaked at pH 8. A linear relationship was observed between cell density and metabolite formation (R2 > 0.996). The rate of metabolite formation was slower in the earlier stages of incubation but then increased. For HO-MPBP, it became constant in the time interval of 2.5-34 h (R2 > 998).