Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of -Hydroxybenzoate and Optimization of Bioproduction Conditions in .

The aromatic compound -hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in cells coexpressing and human NADPH-P450 oxidoreductase () genes as a redox partner. For suitable coexpression of and , we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when expression was induced at 20°C in 2×YT medium in host strain Δ transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and cell concentration), we achieved 90% whole-cell conversion of benzoate. Our data demonstrate that the described novel bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.