Functional Characterization Of CYP716A249 Enzyme In Triterpenoid Saponins Biosynthesis In Polygala Tenuifolia Willd. By Saccharomyces Cerevisiae

Rationales: Polygala tenuifolia,one of the medicinal materials of our country in common use,has the efficacy of tranquilization,glaangal,eliminating sputum and detumescence.Triterpenoid saponin is the main active ingredient of Polygala tenuifolia,and is especially suitable for developing as a new drug with anti-aging and neuroprotective effects.There are two factors limited the development and utilization of Polygala tenuifolia.In the aspect of resources,Polygala tenuifolia was gradually transformed from wild growth to artificial cultivation from the 1980 s onwards.Due to the lack of standardized plant management,it is facing several problems:(1)Polygala has many species.There are42 species and 8 varieties of Polygala plants in China,which are widely distributed in the northeast,north,northwest and central China,Sichuan,etc.And there are many varieties could be used as medicines in ancient times.(2)Polygala tenuifolia has no commercial seeds.(3)Excessive use of fertilizers and pesticides during artificial planting.These made the quality of Polygala tenuifolia uneven and the germplasm gradually degenerate.In the aspect of accessibility,saponins in Polygala tenuifolia are diverse in species,complex in structure,and have many chiral centers in the compound,resulting in the saponin structure more complex derived from a single aglycone.These made it difficult to separate and purify the monomers from plants,and the yield of chemical synthesis is not high.The heterologous synthesis of natural products provides a new idea for the development of triterpenoid saponins.The thinking is to research the key enzymes involved in the triterpenoid saponin biosynthesis pathway of Polygala tenuifolia,and transform the entire biosynthesis pathway to microorganisms to produce the natural products or its precursors.Therefore,it is important to illuminate the key enzymes involved the triterpenoid saponin biosynthesis pathway.In previous studies,we found that CYP716A249 could use ?-amyrin as substrate and to catalyzes the ?-amyrin into oleanolic acid.Therefore,the synthetic pathways of ?-amyrin were constructed in Saccharomyces Cerevisiae by introducing ?-amyrin synthase in this study.In addition,tHMGR and ERG20 genes were overexpressed to increase the precursor supply for improving ?-amyrin production.Then,synthetic pathway of oleanolic acid was constructed in SaccharomycesCerevisiae to confirm the function of later CYP450 involved in the triterpenoid saponin biosynthesis pathway of Polygala tenuifolia.Next,this study analyzed the bioinformatics of CYP716A249 and its mRNA expression levels in different tissues at different growth stages of Polygala tenuifolia to obtain the basic profile of CYP716A249.Finally,the function of CYP716A249 enzyme was confirmed in Saccharomyces Cerevisiae producing?-amyrin.These results laid a scientific foundation for elucidating the biosynthesis pathway of the saponins in Polygala tenuifolia and accelerating the discovery of new drugs related to saponins.Objective: 1.To construct the synthetic pathway of ?-amyrin in Saccharomyces Cerevisiae,which provide the substrate for CYP716A249.2.To construct the synthetic pathways of oleanolic acid in Saccharomyces Cerevisiae.3.To verify the function of CYP716A249.Methods: 1.Overexpression plasmids of tHMGR ? ERG20 and GgbAS were constructed by restriction digestion,in vitro ligation,OE-PCR and homologous recombination.Then transformed overexpression plasmids into yeast cells by LiAc method,to gain the Saccharomyces Cerevisiae producing ?-amyrin.2.Expression plasmid of MtOAS-CPR was constructed by restriction digestion,in vitro ligation and OE-PCR.Transformed the plasmid into yeast cells producing ?-amyrin by LiAc method.Then to detect the oleanolic acid by LC-MS and to get the Saccharomyces Cerevisiae producing oleanolic acid.3.To analyze the nucleotide and protein sequence of CYP716A249 by DNAstar,MEGA 5.1,https://www.ncbi.nlm.nih.gov/orffinder/,http://web.expasy.org/protparam/,https://www.genscript.com/psort.html,http://www.ebi.ac.uk/interpro/search/sequence-search,and http://www.cbs.dtu.dk/services/TMHMM/.And to analyze the expression level of CYP716A249 in the Polygala tenuifolia by RT-qPCR.4.Expression plasmid of PtOAS-CPR was constructed by restriction digestion,in vitro ligation and OE-PCR.Transformed the plasmid into Saccharomyces Cerevisiae producing ?-amyrin by LiAc method.Then to detect the oleanolic acid by LC-MS and to verify the function of CYP716A249.Results: 1.Overexpression plasmids of tHMGR ? ERG20 and GgbAS were constructed and then transformed them into yeast cells to product ?-amyrin.Strain W303-?-amyrin1 was obtained,which produced 1.98 mg/L ?-amyrin.Strain W303-?-amyrin2 was obtained,which produced 4.35 mg/L ?-amyrin.Strain W303-?-amyrin3 was obtained,which produced 10.51 mg/L ?-amyrin.2.Expression plasmids of MtOAS-CPR was constructed and then transformed it into yeast cells.Strain W303-O2 was obtained,which produced 1.30 mg/L oleanolic acid.Then obtaining the Saccharomyces Cerevisiae producing oleanolic acid.3.This research analyzed the sequence and predicted the function of CYP716A249.The results showed that the protein of CYP716A249 is hydrophilic and unstable,and resides mainly in the mitochondria.In Polygala tenuifolia,CYP716A249 is the oleanolic acid synthase that catalyzes the ?-amyrin into oleanolic acid.The results of RT-qPCR showed that the expression level of CYP716A249 were highest in roots of biennial Polygala tenuifolia.4.Expression plasmids of PtOAS-CPR was constructed and then transformed it into yeast cells.And oleanolic acid was detected in the yeast cells.It means that CYP716A249 is the oleanolic acid synthase that using ?-amyrin as substrate and catalyzes the ?-amyrin into oleanolic acid.Conclusion: This research constructs Saccharomyces Cerevisiae producing ?-amyrin and oleanolic acid respectively,and verifies the function of CYP716A249.These results lay a scientific foundation for elucidating the biosynthesis pathway of the saponins in Polygala tenuifolia to realize artificial biosynthesis of saponins,and for accelerating the discovery of new drugs related to saponins.