Abstract
The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2(mut) whole-cell biocatalyst with an outstanding 3'-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2(mut) was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2(mut) (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2(mut) (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3',4'-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.