Study On The Function Of Polygala Tenuifolia CYP88A85 In Saccharomyces Cerevisiae

Selection basis: Polygala tenuifolia is one of the authentic medicinal materials in Shanxi Province,and the demand in the country is large.Polygala saponins are the main active ingredient for its nootropic effect.It belongs to oleanane type pentacyclic triterpene saponin.Its structure is complex.The saponin is expensive and difficult to obtain,which restricts the development of innovative drugs of this type.Saccharomyces cerevisiae grows faster in a suitable environment,especially in large liquid fermentation tanks.Its DNA gene sequence is known,so it is easy to edit genes,and the yeast has the precursor compounds needed to synthesize terpenes(2,3-epoxy squalene),which gives yeast a considerable advantage in synthetic biology research.The heterogeneous synthesis of natural products provides new ideas for the development of P.tenuifolia saponins.First of all,the function of the P450s(cytochrome P450 monooxygenases,P450s)was studied with catalyzing the synthesis of P.tenuifolia saponin in P.tenuifolia.Secondly,the enzyme digestion and homologous recombination technology were used to construct the gene expression cassette.And then,introducing gene expression cassette into the microbial cell.Finally,the desired compound can be obtained from the microbial metabolite.The study found that there may be a competitive relationship between CYP88A85 and oleanolic acid synthase(CYP716A249),that is,these two enzymes will compete for β-amyrin as a substrate,and then catalyze the formation of different metabolites by β-amyrin,such as 11-oxo-amyrin and oleanolic acid.Therefore,if the expression of CYP88A85 can be inhibited,more β-amyrin precursor in oleanolic acid metabolism pathway will be provided,which will increase the production of oleanolic acid.To carry out the above research,we must first identify the function of the CYP88A85 gene.CYP88A85 may use β-amyrin as a substrate to catalyze the oxidation of the C-11 position on the β-amyrin,thereby generating 11-oxo-amyrin.Therefore,the bioinformatics of CYP88A85 was first analyzed,and then the m RNA expression levels in the roots,stems,leaves,and flowers of 1-3 years old P.tenuifolia were analyzed,and the expression of CYP88A85 in different years and different tissues of P.tenuifolia can be obtained in this study.Then,using the S.cerevisiae produced β-amyrin by the research team as the chassis cells,the S.cerevisiae strain producing 11-oxo-amyrin was obtained by introducing the exogenous Pt CYP88A85 gene,which provided a platform for the functional identification of Pt CYP88A85.The results of this study will further promote the elucidation of the biosynthetic pathway of P.tenuifolia saponins and lay the foundation for the development and utilization of P.tenuifolia saponins.Purposes:1.To predict the function of CYP88A85 by bioinformatics analysis;2.To conduct the time and space expression analysis of CYP88A85 in P.tenuifolia,and obtain the m RNA expression profile of CYP88A85 in the roots,stems,leaves and flowers of 1-3 years P.tenuifolia;3.To study the function of Pt CYP88A85 in S.cerevisiae,and provide a theoretical basis for the in-depth understanding of the metabolic pathway for oleanolic acid from β-amyrin.Methods:1.Use genetic analysis related software and online bioinformatics analysis tools to perform bioinformatics analysis on the nucleotide sequence and amino acid sequence of CYP88A85 gene in P.tenuifolia.2.Detect the m RNA expression level of CYP88A85 gene at different development time(1-3 years)and different tissues(roots,stems,leaves,flowers)of P.tenuifolia by reverse transcription quantitative PCR(RT-q PCR)technology.3.Build CPR(Cytochrome P450 reductase,cytochrome P450 reductase)gene expression cassette based on enzyme digestion technology,use overlap extension PCR technology to construct Pt CYP88A85 gene expression cassette,and then transfer these two gene expression cassettes to S.cerevisiae strains producing β-amyrin by using lithium acetate conversion method,thereby obtaining the S.cerevisiae strain of 11-oxo-arsinol,detect the metabolite of the microorganism by gas chromatography mass spectrometry(GC-MS),and finally identify the gene function of CYP88A85.Results:1.The results of bioinformatics analysis of the nucleotides and amino acids of the target gene CYP88A85 showed that: CYP88A85 is a hydrophilic protein located in the mitochondria,has instability,and may catalyze the production of 11-oxo-amyrin from β-amyrin in the P.tenuifolia saponins synthesis pathway.2.The results of RT-q PCR experiments showed that CYP88A85 was the highest expressed in 2-year-old roots of P.tenuifolia.3.The CYP88A85-CPR expression vector was constructed and transferred into S.cerevisiae to produce 11-oxo-amyrin.Conclusion:In this study,S.cerevisiae producing β-amyrin was used as the chassis cell to conduct a functional study on 11-oxo-amyrin synthase-CYP88A85 in P.tenuifolia,and to construct the S.cerevisiae strain producing 11-oxo-amyrin which will promote the elucidation of the biosynthetic pathway of P.tenuifolia saponins and lay the foundation for the development and utilization of Polygala saponins.