Regioselectivity-driven evolution of CYP102D1 for improved synthesis of 3'-ortho-dihydroxyisoflavone

Enzyme and Microbial Technology
Kwon-Young ChoiByung-Gee Kim

Abstract

Daidzein is a major component of isoflavones, and its hydroxylated forms are valuable phytochemicals with anti-cancer and anti-oxidant activity. Due to the limitations of chemical synthesis of these hydroxylated structures, alternative enzymatic synthesis has been attempted. Previously, several protein-engineering approaches using CYP102D1 were investigated; these produced mutants with daidzein hydroxylation activity and regioselectivity through rational design (F96V/M246I) and saturation mutagenesis (A273H/G274E/T277G). However, the generated mutants have low regioselectivity (F96V/M246I) or low hydroxylation activity (A273H/G274E/T277G). Here, we characterized mutants capable of catalyzing C3'-specific daidzein hydroxylation with enhanced hydroxylation activity and regioselectivity. In order to obtain regioselectivity toward the daidzein C3'-position, site-saturation mutagenesis on the substrate-binding region of CYP102D1 F96V/M246I was investigated. A high-throughput screening assay was then performed, based on O-dealkylation activity against the daidzein analog substrate 4'-O-methyl-daidzein. This resulted in a mutant with more than 23-fold improved hydroxylation activity (55.6±17.9μM(-1)min(-1), or 48.4mg/L titer) and regi...Continue Reading

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Citations

Nov 11, 2017·Applied Microbiology and Biotechnology·Eunok JungByung-Gee Kim
Sep 6, 2018·FEMS Microbiology Letters·Pyung-Gang LeeByung-Gee Kim

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