A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

Area of Science:

• Plant biochemistry and molecular biology
• Metabolic engineering
• Enzyme catalysis

Background:

• Flavonoid glycosides are important plant metabolites with diverse biological activities.
• Understanding their biosynthesis pathways is crucial for improving crop quality and yield.
• Safflower (Carthamus tinctorius) is a valuable crop, but its metabolic pathways for secondary metabolites are not fully elucidated.

Purpose of the Study:

• To identify and characterize a key glycosyltransferase involved in flavonoid glycoside biosynthesis in safflower.
• To explore the potential of this enzyme as a biocatalyst for synthesizing flavonoid glycosides.
• To investigate the in vivo function of the identified enzyme in safflower.

Main Methods:

• Identification and cloning of the glycosyltransferase gene (CtUGT4) from safflower.
• Heterologous expression and purification of the CtUGT4 fusion protein in Escherichia coli.
• In vitro enzymatic assays using purified recombinant CtUGT4 with various flavonoid substrates.
• Transient expression of CtUGT4 in tobacco to confirm enzyme activity.
• Overexpression of CtUGT4 in safflower to assess its in vivo function and impact on metabolite content.

Main Results:

• The identified glycosyltransferase, CtUGT4, exhibited flavonoid-O-glycosyltransferase activity.
• Recombinant CtUGT4 catalyzed the formation of quercetin-7-O-glucoside and kaempferol-3-O-glucoside in vitro.
• Enzyme activity was confirmed in vivo through transient expression in tobacco, producing kaempferol-3-O-glucoside and quercetin-3-O-glucoside.
• Overexpression of CtUGT4 in safflower significantly increased quercetin-3-O-rutinoside content and tended to increase quercetin-3-O-glucoside.

Conclusions:

• Safflower CtUGT4 functions as a flavonoid-O-glycosyltransferase, demonstrating substrate specificity and regioselectivity.
• The study provides valuable insights into flavonoid glycoside biosynthesis in safflower.
• CtUGT4 serves as a potential biocatalyst for flavonoid glycoside synthesis and a target for metabolic engineering in safflower cultivation.