Functional expression of cytochrome P450 in Escherichia coli: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l−1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.