Characterization of the 2,6-Dimethylphenol Monooxygenase MpdAB and Evaluation of Its Potential in Vitamin E Precursor Synthesis

Abstract

Although the microbial degradation of the six isomers of dimethylphenol has been extensively studied, the genetic and biochemical mechanisms of 2,6-DMP degradation remain unclear. This study identified the genes responsible for the initial step in the 2,6-DMP catabolic pathway in M. neoaurum B5-4.

Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone

“…Construction of Recombinant Plasmids. For site-directed mutagenesis of MpdA, mpdA mutant fragments were amplified from plasmid pET28a-mpdA 20 via overlap PCR with primers carrying point mutations (Table S2). The resulting fragments were fused into pET-28a(+) using a ClonExpress II One Step cloning kit (Vazyme, Nanjing, China).…”

“…The expression and purification of MpdB and MpdA were performed as described previously. 20 Specifically, E. coli BL21(DE3) containing pET28a-mpdB was initiated by the addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the optical density of the culture at 600 nm (OD 600 ) reached 0.6 and was incubated for an additional 12 h at 150 rpm and 16 °C. For the expression of MpdA, 0.5 mg•mL −1 L-arabinose and 0.2 mM IPTG were used to induce the expression of chaperone protein and recombinant MpdA.…”

“…Kinetic data were measured by nonlinear regression analysis with the Michaelis−Menten equation according to our previous work. 20 One unit of enzyme activity was defined as the amount of enzyme required to catalyze the consumption of 1 μmol of 2,3,6-TMP per min at 30 °C. All data were derived from three independent experiments.…”

“…19 Furthermore, in our previous study, a twocomponent flavin-dependent monooxygenase MpdAB from Mycobacterium neoaurum B5-4 was found that could quantitively transform 2,3,6-TMP to 2,3,5-TMHQ. 20 In this system, the function of the reductase MpdB is to reduce the oxidized FAD and provide the reduced FADH 2 to the oxygenase MpdA. Then, FADH 2 is used by MpdA to activate molecular oxygen for substrate oxidation (Figure 1C).…”

“…Then, FADH 2 is used by MpdA to activate molecular oxygen for substrate oxidation (Figure 1C). 20 However, the poor activity of MpdA and cytotoxicity of 2,3,6-TMP limited the yield of 2,3,5-TMHQ. Therefore, suitable enzymes or microbes are urgently needed to produce 2,3,5-TMHQ through biological or biological−chemical pathways to accelerate the green and low-cost synthesis of vitamin E. This work aims to construct an improved whole-cell biocatalyst for the production of 2,3,5-TMHQ by enhancing the catalytic ability of enzymes and cell tolerance.…”