Biochemical Characterization of a Flavone Synthase I from Daucus carota and its Application for Bioconversion of Flavanones to Flavones.

In this study, we studied the biochemical characterization of flavone synthase I from Daucus carota (DcFNS I) and applied it with flavonoid 6-hydroxylase from Scutellaria baicalensis (SbCYP) to convert flavanones to flavones. The recombinant DcFNS I was expressed in the form of the glutathione-S-transferase fusion protein. Rather than taxifolin, naringenin, pinocembrin, and eriodictyol were accepted as substrates. The optimal temperature and pH for reaction in vitro were 35 °C and 7.5, respectively, and 2-oxoglutarate was essential in the assay system. Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) were not substitutes for Fe(2+). EDTA and pyruvic acid inhibited the activity, except for Fe(3+). Kinetic analysis revealed that the V(max) and k(cat) values of the recombinant DcFNS I against naringenin were 0.183 nmol mg(-1) s(-1) and 0.0121 s(-1), and 0.175 nmol mg(-1) s(-1) and 0.0116 s(-1) against pinocembrin. However, the recombinant DcFNS I had a higher affinity for naringenin than pinocembrin, with k(M) values for each of 0.076 mM and 0.174 mM respectively. Thus, it catalyzed naringenin more efficiently than pinocembrin. Subsequently, using an Escherichia coli and Saccharomyces cerevisiae co-culture system, we successfully converted naringenin and pinocembrin to scutellarein and baicalein respectively. In a synthetic complete medium, the titers of scutellarein and baicalein reached 5.63 mg/L and 0.78 mg/L from 200 mg/L precursors.