Research To The Biosynthesis Of Ursolic Acid And Oleanolic Acid By Saccharomyces Cerevisiae

Ursolic acid and oleanolic acid are isomers and are pentacyclic triterpenoids with various biological activities.Ursolic acid has many biological effects such as anti-inflammatory,antibacterial,anti-ulcer,anti-oxidation and anti-diabetes,whichhas been widely used in medicine and cosmetics.The most important pharmacological activity of oleanolic acid is its hepatoprotective effect.In addition,studies have shown that the combination of ursolic acid and oleanolic acid has a stronger ability inhibitingthe proliferation of malignant cells than each of them.In this paper,engineered strains of Saccharomyces cerevisiae,which can synthesize ursolic acid and oleanolic acid,were constructed.Shake flasks fermentation and 5L fermenter fed-batch fermentation were studied further to increase the yield of ursolic acid and oleanolic acid.First of all,the expression plasmid pXP320-CrMAS,which is a multifunctional amyrin synthase,was constructed.The expression plasmid was transformed into Saccharomyces cerevisiae WTE to obtain the strain ScLCZ02 that produces ?-amyrin and ?-amyrin.The GAL1p-CrMAS-CYC1t-TEF1p-CaERG1-ADH1 t expression module was introduced into rDNA site of strain WTE to obtain strain ScLCZ08.The initial glucose concentration and galactose-induced concentration were optimized by shake flask fermentation.The yields of ?-amyrin and ?-amyrin were increased to97.31 mg/L and 24.95 mg/L,respectively.Secondly,we tried to combine two cytochrome P450 enzymes,MtOAS and CrOAS,with AtCPR1 and LjCPR,to obtain four kinds of strains ScLCZ11-14 which could synthesize different proportions of ursolic acid and oleanolic acid.ScLCZ11,the highest yielding strain,was able to synthesize25.85 mg/L ursolic acid and 31.41mg/Loleanolic acid.Strain ScLCZ13 is more capable of producing ursolic acid.The yield of ursolic acid was 23.37 mg/L,accounting for 48.98% of the total yield,while it was only 45.14% of the total yield in ScLCZ11 strain.Then,four macroporous resins D101,D3520,HPD-100 and X-5 were used for adsorption and desorption experiments of ursolic acid and oleanolic acid.X-5,which was the optimal macroporous resin,was selected for the study of product fermentationcoupling with separation in shake flask.The results show that the addition of resin will inhibit the growth of yeast cell and product synthesis,andon-line separation need to be further studied.Finally,employing two-stage fermentation process with feeding glucose and ethanol,the yields of ursolic acid and oleanolic acid reached 123.27 mg/L and 155.58mg/L in 5L fermenter,which is 4.77 times and 4.95 times higher than the original strain,respectively.