Cloning And Expression Of Daidzein C-glycosylation Related Enzyme Genes

Pueraria lobata is a traditional Chinese medicine in China,which is widely used in clinical practice and has good curative effect on cardiovascular and cerebrovascular diseases,diabetes,liver diseases and other diseases.Isoflavones are the secondary metabolites with the highest content in Kudzu vine.Pueraria lobata is a plant medicine with anti-inflammatory,anti-tumor,and antioxidant effects.Puerarin is a plant with great medicinal value.Its unique feature is that its glycosylation is connected to the parent ring through C-C bonds,while most isoflavone glycosides are connected to the aglycone ring through C-O bonds.There are two corresponding UGTs in Pueraria lobata,namely CGT(Carboglycosyl transfer)and OGT(Oxyglycosyl transfer).Glycosyltransfers(GTs)are a natural substance that is a key enzyme in the metabolic pathway called glycosyltransferases,which can transfer UDP activated glycosides into biological macromolecules.UGT can convert flavonoids into isoflavone glycosides,playing a crucial role in the synthesis,transportation,and storage of flavonoids.In this study,a PIUGT43 gene sequence with a length of 1410 bp was obtained by PCR amplification using pueraria c DNA sequence as template.Bioinformatics analysis showed that the protein encoded by PIUGT43 gene was a nontransmembrane protein with a molecular weight of about 52.4k Da,a theoretical isoelectric point of 5.83,a negative charge residue(Asp + Glu)of 49 and a positive charge residue(Arg + Lys)of 40.The instability coefficient was 42.31,which was an unstable hydrophilic protein.PIUGT43 gene was cloned into Escherichia coli expression vector p ET-28 a and p ESC-TRP yeast expression vector,and was successfully expressed in BL21 and Chessam saccharomyces cerevisiae.HPLC assay showed that PIUGT43 enzyme had certain activity and could catalyze the production of puerarin from daidzein and Uridine diphosphate glucose(UDPG).In order to break through the technical bottleneck in the production of puerarin,this experiment successfully constructed a new type of 8-C-glucosyltransferase producing yeast strain.The project team established a new method for microbial heterologous synthesis of puerarin,which laid a solid foundation for the industrial development and application of puerarin.