Development of Colorimetric HTS Assay of Cytochrome P450 for ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity

A current challenge in high‐throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid‐agar plate‐based HTS assay for screening ortho‐specific hydroxylation of daidzein by sensing formaldehyde generated from the O‐dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde‐specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho‐carbon position to COH correlates with a linear relationship to O‐dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4′,7‐dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B‐ring, was examined for O‐dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O‐dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A‐ring inactivated in order to find enhanced 3′‐regioselectiviy). The whole‐cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L−1 production titer and greatly increased 3′‐regioselectiviy (3′/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions. High‐speed activity: A general HTS assay method for ortho‐specific hydroxylation by P450 enzymes is presented. In order to screen for P450 variants with higher hydroxylation activities, an indirect HTS assay method to sense aldehyde molecules generated from O‐dealkylation reaction of chemically permethylated substrate was developed.