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

Authors

  • Dr. Kwon-Young Choi,

    1. School of Chemical and Biological Engineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul (South Korea)
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  • Eun-Ok Jung,

    1. School of Chemical and Biological Engineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul (South Korea)
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  • Prof. Dr. Hyungdon Yun,

    1. School of Biotechnology, Yeungnam University, 280 Daehak-ro, 712-749 Gyeongsan, Gyeongbuk (South Korea)
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  • Prof. Dr. Yung-Hun Yang,

    1. Department of Microbial Engineering, College of Engineering, Konkuk University, 120 Neungdong-ro, 143-701 Seoul (South Korea)
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  • Prof. Dr. Romas J. Kazlauskas,

    1. Molecular Biology & Biophysics and BioTechnology Institute, Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108 (USA)
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  • Prof. Dr. Byung-Gee Kim

    Corresponding author
    1. School of Chemical and Biological Engineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul (South Korea)
    2. Institute of Bioengineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul (South Korea)
    • School of Chemical and Biological Engineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul (South Korea)===

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Abstract

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[BOND]OH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding C[BOND]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.

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