Preliminary Exploration On Production Of Salidroside By Saccharomyces Cerevisiae

Salidroside is an effective active ingredient of Rhodiola plants,which has beneficial effects such as anti-inflammatory,anti-radiation,anti-oxidation,and promotion of cancer cell apoptosis.Extraction salidroside from plant is hard to carry out large-scale industrial production due to limited resources,low content,and difficult extraction.The chemical synthesis method is not suitable for salidroside production because of the large consume of organic solvents,the large amounts of hazardous waste,and environmental pollution.In this experiment,aiming at increasing the amount of salidroside synthesis in Saccharomyces cerevisiae,two parts of increasing the amount of tyrosol synthesis in S.cerevisiae and enhancing the tyrosol conversion rate were investigated.In addiction,an in vitro enzyme synthesis system was constructed to synthesize salidroside through extracorporeal reaction.In order to increase the amount of tyrosol synthesis in S.cerevisiae,we studied three strains of prephenate dehydrogenase(PDHs)(EC.1.3.1.12)from Streptococcus mutans UA159,Novosphingobium sp.PP1Y and Escherichia coli after the introduction of the titer of tyrosol synthesis,and the transcription level of exogenous PDH.The experiment found that although the exogenous PDH transcription level was higher than that of the internal reference gene,it failed to increase the production of tyrosol.The tyrosol titer of the control strain B2-1 and the transformants B2-1Sm,B2-1Np,and B2-lEc introduced with the exogenous PDH plasmid were 100.80 mg/L,80.82 mg/L,85.30 mg/L,and 82.20 mg/L,respectively.Salidroside requires the participation of glycosyltransferase for biosynthesis with tyrosol as a substrate.It is synthesized by transferring the glycosyl of the donor to the alcohol hydroxyl group of tyrosol by glycosyltransferase.Uridine diphosphate glucosyltransferases(UGTs)from Arabidopsis thaliana and Rhodiola sachalinensis:UGT85A1,UGT73B6M264K/V36IT/T3731/G376S was introduced to Saccharomyces cerevisiae after metabolic engineering transformation,and Saccharomyces cerevisiae BH1 and BH2 capable of synthesizing salidroside were constructed.Furthermore,the culture conditions were investigated to increase the titer of salidroside.When the addition of tyrosol was 5 mM,53.54 mg/L salidroside was synthesized under the culture conditions of 3%glucose in S.cerevisiae BH1 introduced into UGT85A1,and BH2 introduced into UGT73B6M264K/V361T/T3731/G376 obtained 25.33 mg/L salidroside.When glucose was 2%,with the addition of 10 mM tyrosol,BH1 synthesized 28.15 mg/L salidroside.Moreover,we constructed an in vitro enzymic reaction to synthesize salidroside by using sucrose and tyrosol as substrates and catalyzed by Arabidopsis thaliana UDP-glucuronosyltransferases UGT85A1 and Arabidopsis thaliana sucrose synthase SUS1.When the substrate sucrose and tyrosol were supplied in excess,1334.1 mg/L salidroside was obtained by adding 100 mg/L of uridine-5'-diphosphate glucose(UDP-G)and 10 mM Mg2+.