Construction Of Saccharomyces Cerevisiae PtP450s Research System Based On Homologous Recombination

Rationales: Polygala tenuifolia is an important medicinal material in China,and it is also a local medicinal material in Shanxi.As one of the main active ingredients of Polygala tenuifolia,polygala saponins have pharmacological effects such as anti-dementia,memory impairment and brain protection.However,since saponin belongs to the oleanane-pe pentacyclic triterpenoid saponin,its aglycones are diverse in variety and complex in structure,and have multiple chiral centers,resulting in a more complex saponin structure derived from a single aglycone.There are more than50 kinds of saponins derived from only the senegenin.Direct extraction of polygala saponins from Polygala tenuifolia faces many disadvantages such as low content of active ingredients,limited resources of medicinal materials,and difficulty in purification.However,through the full chemical synthesis,the steps are cumbersome due to the multi-step purification,and it is difficult to guarantee a higher yield.The method of tissue cell culture obtains saponin,and it faces a long and complicated operation cycle,and it is difficult to achieve large-scale industrial production.The emergence of synthetic biology,especially the heterologous biosynthesis of terpenoids using Saccharomyces cerevisiae,provides a new method for the acquisition of saponins.S.cerevisiae grows fast,it is easy to be genetically engineered,is suitable for large-scale culture,and is easy to control industrially.S.cerevisiae can provide a natural synthetic steroid precursor 2,3-oxidized squalene,which facilitates the biosynthesis of steroids by heterologous biosynthesis.Our group has previously verified a cytochrome P450 monooxygenase CYP716A249(cytochrome P450 monooxygenase CYP716A249,referred to as CYP716A249 or Pt OAS)that synthesizes the saponin precursor oleanolic acid.By introducing Glycyrrhiza glabra ?-AS(Ggb AS,Genbank: AB037203.1),Lotus japonicus CPR(Lj CPR,Genbank: AB433810)and Medicago truncatula OAS(Mt OAS,Genbank: FN995113.1),CYP716A249(Pt OAS,Genbank: KY385302.1)in Saccharomyces cerevisiae W303 a.The plasmid p RS304-ADH1p-Pt OAS-ADH1t-ALA1p-Lj CPR-ALA1 t was constructed by doublerestriction enzyme ligation and it was successfully transferred into S.cerevisiae producing ?-amyrin.The content of oleanolic acid was 1.32 mg/L by metabolite detection,and the content of oleanolic acid was decreased by overexpressing the upstream key enzyme gene ERG20,which was 0.13 mg/L.The reason may be that too much plasmid was transferred into the S.cerevisiae,which makes the S.cerevisiae overloaded,and it was difficult to provide energy for the subsequent exogenously introduced plasmid,resulting in a decrease in yield.Therefore,based on the efficient DNA homologous recombination ability of Saccharomyces cerevisiae,this study regulates the endogenous genes of S.cerevisiae and introduces foreign genes by S.cerevisiae transformation-associated recombination(TAR)technology,construction of S.cerevisiae producing ?-amyrin and oleanolic acid;based on q RT-PCR and GC-MS,UPLC-MS technology analyzes the content of oleanolic acid biosynthesis-related genes and metabolites in different S.cerevisiae engineering strains from m RNA levels and metabolite levels;by comparing restriction enzyme ligation(strain prefix RL-)and homologous recombination(strain prefix HR-)engineered the effect of S.cerevisiae on metabolite accumulation and analyzed the reasons for increasing metabolite yield.The above studies provide a new method for the biosynthesis of Polygala saponins in S.cerevisiae,and lay a foundation for the construction of the S.cerevisiae P450 s in the body function research system.Objective:1.The S.cerevisiae HR-?-AS producing ?-amyrin was constructed by homologous recombination technology,which provided a substrate for the subsequent transfer of gene expression cassettes such as OAS.2.The Lj CPR was introduced to construct the S.cerevisiae engineering strain HR-Lj CPR which provides electrons for subsequent P450 s.3.Construction of homologous recombinant S.cerevisiae HR-Mt OAS and HR-Pt OAS,regulation of endogenous gene ERG20,construction of S.cerevisiae HR-Mt OAS-ERG20 and HR-Pt OAS-ERG20.4.Analyze the effect of the strain constructed by homologous recombinationVII technology and the restriction enzyme ligation method on the content of oleanolic acid.Methods:1.Amplification of homologous recombination expression elements of the gene of interest using the principle of gene homologous recombination technology.2.The homologous recombinant expression elements of the above target gene,including the selection markers,were sequentially transferred into S.cerevisiae W303 a to construct different S.cerevisiae engineering strains.3.Quantify related genes and metabolites by q RT-PCR,GC-MS and LC-MS techniques,and analyze the effects of different construction methods on target metabolite yield.Results:1.Amplification of homologous recombination expression elements of five genes(Gg?-AS,Lj CPR,Pt OAS,Mt OAS and ERG20)using the principle of homologous recombination;2.Successfully construct the S.cerevisiae HR-?-AS producing ?-amyrin,S.cerevisiae HR-Lj CPR providing electronics for P450 s,S.cerevisiae HR-Pt OAS and HR-Mt OAS producing oleanolic acid,HR-Pt OAS-ERG20 and HR-Mt OAS-ERG20,which regulate endogenous genes and increase oleanolic acid production.3.The m RNA expression levels of ?-AS and OAS were determined by q RT-PCR.The results showed that the m RNA expression levels of the OAS gene were higher in the strains constructed by homologous recombination than those constructed by restriction enzyme ligation.After the addition of the ERG20 gene,except for the m RNA expression level of OAS in RL-Pt OAS strain,the m RNA expression levels of?-AS and OAS genes in the strains constructed by homologous recombination and restriction enzyme ligation were higher than those previously expressed.4.The accumulation of oleanolic acid in the metabolite of S.cerevisiae was compared by enzyme digestion an d homologous recombination.The results showed that the content of ?-amyrin in HR-Mt OAS was 4.74 mg/L,which was is 3.5 times higher than that in RL-Mt OAS,the content of oleanolic acid is 10.98 mg/L,which is13 times higher than that of RL-Mt OAS.The content of ?-amyrin in HR-Pt OAS is 2.4times higher than that of RL-Pt OAS,the oleanolic acid content was 20 times higher than that of RL-Pt OAS.After increasing the precursor supply of ERG20,the content of ?-amyrin in HR-Mt OAS-ERG20 was 4.85 mg/L,which was 2.2 times higher than that in RL-Mt OAS-ERG20,the oleanolic acid content was 20.44 mg/L,which was 10 times higher than that of RL-Mt OAS-ERG20.The content of ?-amyrin in HR-Pt OAS-ERG20 was 3.92 mg/L,which was 2.2 times lower than that of RL-Pt OAS-ERG20,and the oleanolic acid content was 15.34 mg/L,which was 100 higher than that of RL-Pt OAS-ERG20.Most importantly,the content of oleanolic acid did not decrease after the introduction of ERG20 by homologous recombination techniques.This result indicates that homologous recombination is a more suitable method for transforming Saccharomyces cerevisiae than the restriction enzyme digestion to obtain natural products.Conclusion:This study utilizes homologous recombination technology to engineer S.cerevisiae synthesize the downstream key enzyme genes of the polygala saponins,providing a new method for heterologous biosynthesis of steroids in S.cerevisiae.It provides a basis for subsequent functional identification of P450 s associated with polygala saponin biosynthesis in S.cerevisiae.