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

Abstract

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.

Genomic and Transcriptomic Study for Screening Genes Involved in the Limonene Biotransformation of Penicillium digitatum DSM 62840

“…CYP53A15 is correlated to detoxification and degradation of benzoate, and is capable of hydroxylating benzoate to 4-hydroxybenzoate. It can be further metabolized by the β-ketoadipate pathway to generate intermediates which will enter into the TCA cycle (Tamaki et al, 2018). Benzoate is essential for the metabolism pathways of aromatic compounds, and could inhibit fungal growth.…”

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli

“…Additionally, this type of strategy has also been successfully used to express this type of enzymes in E. coli [ 56 ]. The truncation or replacement of CYP450 and CPR N -terminal regions led to an increase in CYP450 and CPR solubility and expression [ 57 , 58 ]. N -terminal has been frequently replaced by Met-Ala (as previously discussed) [ 58 , 59 ] or by a ‘universal’ N -terminal sequence (e.g., 8RP, MALLLAVF; 2C3, MAKKTSSKGK; 2B1, MAKKTSSKGKLPPG(PS)) [ 58 , 59 , 60 , 61 , 62 , 63 ].…”

“…The truncation or replacement of CYP450 and CPR N -terminal regions led to an increase in CYP450 and CPR solubility and expression [ 57 , 58 ]. N -terminal has been frequently replaced by Met-Ala (as previously discussed) [ 58 , 59 ] or by a ‘universal’ N -terminal sequence (e.g., 8RP, MALLLAVF; 2C3, MAKKTSSKGK; 2B1, MAKKTSSKGKLPPG(PS)) [ 58 , 59 , 60 , 61 , 62 , 63 ]. More complex N -terminal sequences containing 84–214 aa have also been used (e.g., 28-tag, Sumo, MBP) [ 59 , 61 ].…”