Functional Study Of The Key Enzyme P450 (C-2α ) In The Biosynthesis Of Polygonate Saponins

Rationales:Polygala tenuifolia Willd,the genus Polygala,has a wide range of medicinal values and a long history,and has a greater value for medicinal research and development and utilization.Polygala saponin plays an important role in expectorant and cough suppressant and educational.The cost and low yield of Polygala saponin by traditional chemical synthesis or direct extraction are high.With the development of synthetic biology,it has become possible to Polygala saponin by means of biosynthesis.Among them,precursor supply and intermediate construction have been well studied,and the terpene formation part of the biosynthetic pathway of Polygala saponins is more complex.Research progress has been very slow.In the next step,we have focused on the terpene formation.Cytochrome P450 monooxygenase is responsible for the change of oleanolic acid to Polygala saponin through multi-site catalysis,mainly focusing on C-2α,C-2β,C-23 and C-27 sites.This study focuses on the C-2α site,and after observing the change in chemical formula.oleanolic acid alone undergoes hydroxylation at C-2α site and other sites remain unchanged,according to the change in chemical formula is to produce maslinic acid,This study focused on the production of Maslinic acid catalyzed by oleanolic acid via the C-2α site.Our second and third generation transcriptome databases were combined with the National Center of Biotechnology Information,NCBI P.tenuifolia Database,Database of plant P450 s established by Tianjin Institute of Industrial Biology(Plant Cytochrome P450 Database Database website: https://p450.biodesign.ac.cn/)three constructing phylogenetic trees,for the determination of the specific site,The amino acid sequences of the three databases were subjected to phylogenetic analysis,and the key P450 s enzyme genes of oleanolic acid to Polygala saponin were predicted using FPKM(Fragments Per Kilobase Million,representing fragments per kilobase of transcription per million mapped reads)values in the group’s second and third generation transcriptome database.Quantitative Real-time PCR(q RT-PCR)technology was used to quantify the expression of m RNA of key genes screened in the transcriptome database of the second and third generation of P.tenuifolia to support the screening of key enzyme genes of Polygala saponin in the biosynthetic pathway,and finally selected key P450 s enzyme CYP92A143 that act at the C-2αposition And CYP92A6 key enzymes.A series of analyses were conducted around CYP92A6 and CYP92A143 with bioinformatics related.The overlapping extension polymerase chain reaction(OE-PCR)was used to construct gene expression cassettes for both(delta-up-HIS,ADH1p+CYP92A6+ADH1t,ADH1p+CYP92A143+ ADH1 t and delta-down),and the expression elements were directly transformed into Saccharomyces cerevisiae by ultra performance liquid chromatography-mass spectrometry(UPLC-MS),thus initially probing the function in heterologous expression in Saccharomyces cerevisiae.The results showed that CYP92A6 and CYP92A143 successfully detected maslinic acid,substances in vivo and in microsomal reactions in vitro,suggesting that these two genes act on the oleanolic acid substrate C-2α site to produce a catalytic effect.Objective:1.The phylogenetic tree was constructed using the transcriptome database of the second and third generation of P.tenuifolia,the NCBI P.tenuifolia database,and the Plant P450 s database.β-AS and screened P450 s were analyzed,mainly by using SPSS for normality test and pearson analysis.Final screening of candidate genes acting on C-2α;2.two gene expression cassettes(delta-up-HIS,ADH1p+CYP92A6+ADH1t,delta-down,delta-up-HIS,ADH1p+CYP92A143+ ADH1 t and delta-down)transferred into Saccharomyces cerevisiae strains(HR-Mt OAS,HR-CPR).Methods:1.We used MEGA X software to construct a phylogenetic tree of the transcriptome database of the second and third generation of P.tenuifolia,the NCBI P.tenuifolia database combined with the plant P450 s database,and used DNAMAN software for sequence alignment and SMART for related structure-function domain analysis.The FPKM values of the β-AS and the screened P450 s in the transcriptome database of the second and third generation of P.tenuifolia,were tested for normality using SPSS 24.0 software,and the FPKM values of the β-AS and the screened candidate P450 s genes in the transcriptome database of the second and third generation of P.tenuifolia,were subjected to pearson analysis using Grandpad;2.Bioinformatics related websites and software such as Prot-Param tool,Prot Scale,TMHMM Serverv.2.0,Signal P-4.1,Wo LF PSORT,DIpro,SMART,SOPMA,SWISS-MODEL,DNAMAN were used to predict and analyze the structure and biological properties of positive controls pea CYP92A6,P.tenuifolia candidate CYP92A143 proteins by predicting and analyzing their structures and biological properties;3.The genes delta-up,HIS,ADH1 t and delta-down were amplified in Saccharomyces cerevisiae W303 a,the gene ADH1 p was amplified in plasmid PUC57,the genes CYP92A6 and CYP92A143 were amplified in plasmid p UC-GW-Kan and two gene expression cassettes were constructed using OE-PCR(delta-up-HIS,ADH1p+ CYP92A6 + ADH1 t,delta-down,delta-up-HIS,ADH1 p + CYP92A143 + ADH1 t and delta-down)using gene homologous recombination methods and lithium acetate technology and transferred into Saccharomyces cerevisiae strains(HR-Mt OAS,HR-CPR);4.An ultra performance liquid chromatography-mass spectrometry(UPLC-MS)technique was used to detect the in vivo functional assay and in vitro enzymatic activity reaction of CYP92A143 and CYP92A6 for the production of maslinic acid.Result:1.The phylogenetic tree of CYP92A6,CYP92A143,and c130062 g1 was clustered into one piece by using the transcriptome database of the second and third generation of P.tenuifolia,NCBI P.tenuifolia database,and plant P450 s database.The FPKM values of β-AS and the candidate genes of P450 s were compared with the transcriptomic data for pearson analysis,and finally 15 related genes were identified,including c130062 g1.The positive control genes CYP92A6,candidate genes CYP92A143 with the full length of CDS were finally identified as the study subjects;2.Successful prediction and analysis of the structure and biological properties of pea CYP92A6 and P.tenuifolia CYP92A143 proteins using bioinformatics-related web sites and software;3.Gene elements were amplified and gene expression cassettes(delta-up-HIS,ADH1 p + CYP92A6 + ADH1 t,delta-down,delta-up-HIS,ADH1 p + CYP92A143 +ADH1t and delta-down)were constructed using OE-PCR.4.The CYP92A143 and CYP92A6 genes were sequentially transferred into HR-CPR and HR-Mt OAS strains,respectively,and four yeast strains containing CYP92A143 and CYP92A6 were successfully constructed,verified by PCR and sent for sequencing to verify;5.The in vivo and in vitro functional study products were examined using liquid mass spectrometry UPLC-MS,and the results showed that CYP92A6 and CYP92A143 were successfully detected in vivo and in the in vitro microsomal reaction with oleanolic acid.Conclusion:In this study,the candidate genes of C-2α on the biosynthetic pathway of Polygala saponin were screened using the transcriptome sequencing of the transcriptome database of the second and third generation of P.tenuifolia,NCBI P.tenuifolia database,and plant P450 s database as well as the candidate genes of P450 s obtained from the β-AS and screening of transcriptome data,and the candidate genes CYP92A143 and CYP92A6 were analyzed.The relevant genes were amplified,the gene expression cassettes(delta-up-HIS,ADH1p+CYP92A6+ADH1t and delta-down,delta-up-HIS,ADH1 p + CYP92A143 + ADH1 t and delta-down)were constructed according to the gene homologous recombination technique and the gene elements were successfully transferred into Saccharomyces cerevisiae strains were constructed and synthesized.In vitro and in vivo functional validation experiments were performed using liquid mass spectrometry(UPLC-MS).The success of the in vitro and in vivo functional validation experiments Willd provide technical support for the subsequent large-scale functional validation of the genes by the group,and the in vitro preparation of microsomes Willd provide a new platform for in-group functional validation and promote the development of the biosynthetic pathway of Polygala saponin.