A Muti-Substrate Flavonol -glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase UGT4, with flavonoid--glycosyltransferase activity, was identified in safflower. The fusion protein of UGT4 was heterologously expressed in , and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7--glucoside, and kaempferol to form kaempferol-3- in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. UGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3--glucoside, and quercetin to quercetin-3--glucoside. After overexpressing in safflower, the content of quercetin-3--rutinoside in the safflower florets increased significantly, and the content of quercetin-3--glucoside also tended to increase, which preliminarily confirmed the function of UGT4 flavonoid--glycosyltransferase. This work demonstrated the flavonoid--glycosyltransferase function of safflower UGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.