Synthesis Of Salidroside Based On In-situ Regeneration System Of UDP-glucose

Salidroside is an effective component of Rhodiola.It has functions of anti fatigue and antioxidation.Simply relying on wild Rhodiola to extract salidroside can not be able to meet the growing demand,and the artificial cultivation of Rhodiola is restricted by many aspects.The chemical synthesis method may not make it safe because of using organic solvents.The biosynthesis of salidroside has become a popular topic in recent years.However,much attention has been paid to increase the supply of tyrosol and the efficient expression of glucosyltransferase,and the method of UDPG synthesis has often been neglected.This study was based on that uridine diphosphate glucosyltransferase(UGT)catalyzed salidroside synthesis requires the substrate UDPG and produces the by-product UDP,while sucrose synthase(SuSy)can use UDP as a substrate to generate UDPG;Gene fusion method was used to construct fusion enzyme of SuSy and UGT;In situ regeneration system of UDPG was established by the expression of the fusion enzyme in Escherichia coli(E.coli)to achieve biosynthesis of salidroside.First of all,genes of Arabidopsis thaliana sucrose synthase AtSUS1 and Rhodiola sachalinensis uridine diphosphate glucosyltransferase UGT72B14 were cloned;then the sequences of the two cloned genes were optimized according to the host E.coli codon bias.The fusion gene AtSUS1-3a-UGT72B14 was obtained by a linker of three amino acids GSG,which was designed by enzymatic cleavage sites,and then the expression vector pET28a-AtSUS1-3a-UGT72B14 was constructed.The recombinant plasmid was transformed into the host competent cell BL21(DE3);The inducing expression conditions of the fusion protein were optimized.When the cell concentration reached OD600= 0.6,IPTG was added to a final concentration of 0.6 mM.When the temperature was set to 16 ?for 18 h,a large amount of recombinant protein with high purity could be obtained;Finally,the recombinant protein was purified.The operation of the fusion system to obtain salidroside was confirmed by HPLC analysis of the in vitro enzymatic reaction.The reaction solution was detected and the concentration of salidroside was calculated to be 9.17 ?g/ml in accordance with the standard curve.According to the in vitro enzymatic reaction system,the reaction product was relative to the substrate(sucrose + UDP)in the case of tyrosol excess.The conversion rate was 69.26%.It was confirmed that the operation of the fusion system can obtain the salidroside product.