Development and Optimization of an In Vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin.

An in vitro multienzyme synthetic system was developed and optimized to efficiently produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The recombinant proteins were purified through affinity chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase, respectively, followed by development of an in vitro synthetic system for producing kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 0.4% sodium ascorbate, 25 μg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2). When the reaction was carried out at 40 °C for 40-50 min, the yield of kaempferol was 37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%. Overall, this system provides a promising and efficient approach to produce kaempferol economically.