Gene synthesis and expression of Arabidopsis thaliana sucrose synthase At SUS1 in Escherichia coli and application in enzymatic production of rebaudioside A

In this study,the gene encoding sucrose synthase At SUS1 from Arabidopsis thaliana was synthesized by two- step PCR,which was subcloned into the plasmid p ET28a- UGT carrying the gene encoding glycosyltransferase UGT76G1,to construct the recombinant plasmid p EUGT- SUS. Then p EUGT- SUS was transformed into E. coli Rossetta( DE3),and the recombinant strain was cultivated in auto- induction medium for12 h. The collected cells were lysated by ultrasonic and the liquid supernatant was used as the crude enzyme for catalyzing stevioside to form rebaudioside A. The results confirmed that recombinant enzyme At SUS1 was functional expressed in E.coli.In the reaction containing these two enzymes,sucrose and UDP were catalyzed by At SUS1 to generate UDP- glucose,which was supplied to the glycosyltransfer reaction catalyzed by UGT76G1.Influences of different UDP concentration,pH,temperature and reaction time on the enzymatic production of rebaudioside A were investigated. The yield of rebaudioside A from 10 g / L of stevioside was as high as 56.2%when the reaction was carried out with 0.01 mmol / L UDP at pH7.2 and 35 ℃ for 12 h.This study laid a foundation for building an economic and efficiently enzymatic production of rebaudioside A.