Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea.
J Microbiol Biotechnol. 2009 Dec;19(12):1612-6. doi: 10.4014/jmb.0905.05043.
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.
异黄酮是一类植物雌激素。异黄酮合酶（IFS）负责将柚皮素转化为染料木黄酮。IFS 是细胞色素 P450（CYP），其活性需要细胞色素 P450 还原酶（CPR）。此外，大多数细胞色素 P450 都具有膜结合域，这使得它们难以在大肠杆菌中表达。为了解决这些问题，我们在去除了 RCIFS 和 RCPR 中的膜结合域后，构建了红三叶草 IFS（RCIFS）和来自水稻的 CPR（RCPR）的框内融合。所得融合基因 RCIFS-RCPR 在大肠杆菌中表达。使用该大肠杆菌转化体证实了柚皮素向染料木黄酮的转化。在优化了生物转化的培养基和细胞密度后，可从 80 μM 的柚皮素生成 60 μM 的染料木黄酮。这种融合蛋白方法可能适用于在大肠杆菌中表达其他 P450。
