Production of Genistein from Naringenin Using Escherichia coli Containing Isoflavone Synthase-Cytochrome P450 Reductase Fusion Protein

Isoflavonoids are a class of phytoestroegens. Isoflavonone synthase (IFS) is responsible for the conversion of naringenin to genistein. IFS is a cytochrome P450 (CYP), and requires cytochrome P450 reductase (CPR) for its activity. Additionally, the majority of cytochrome P450s harbor a membrane binding domain, making them difficult to express in Escherichia coli. In order to resolve these issues, we constructed an in-frame fusion of the IFS from red clover (RCIFS) and CPR from rice (RCPR) after removing the membrane binding domain from RCIFS and RCPR reductase. The resultant fusion gene, RCIFS-RCPR, was expressed in E. coli. The conversion of naringenin into genistein was confirmed using this E. coli transformant. Following the optimization of the medium and cell density for biotransformation, 60 microM of genistein could be generated from 80 microM of naringenin. This fusion protein approach might be applicable to express other P450s in E. coli.