Cloning And In-vitro Functional Study Of Genes Involving In Cucurbitacin Ⅱa Biosynthesis

Cucurbitacin Ⅱa(Cu Ⅱa)is the main active component of Hemsleya in Cucurbitaceae.It has anti-inflammatory,anti-HIV and anti-tumor activities.Because of its good antibacterial activity and no drug resistance,Cu Ⅱa also has an excellent clinical application prospect.However,the contents of Cu Ⅱa in Hemsleya are low,and the Hemsleya medicinal plants are in short supply.It is also very difficult for chemical synthesis.Therefore,it is urgent to find a new way to get Cu Ⅱa.Recently,the synthetic biology method is expected to be a new way of thinking to get Cu Ⅱa.The biosynthetic pathway of tetracyclic triterpenoids has been extensively studied and developed in various Cucurbitaceae medicinal plants.However,the biosynthetic pathway of Cu Ⅱa in Hemsleya remains unclear,especially squalene epoxidase(SE)and recombinant oxidosqualene cyclase(OSC),including cycloartenol synthase(CAS)and beta-amyrin synthase(β-AS).These enzymes catalyze the formation of critical intermediate precursors and complete further chemical structure modification.Therefore,in-depth research on the genes of enzymes related to the biosynthesis of Cu Ⅱa will lay a foundation for constructing the complete biosynthesis pathway.At the same time,it will provide a basis for the realization of synthetic biology to produce Cu Ⅱa and then alleviate the shortage of Hemsleya resources.In this paper,Hemsleya macrosperma was used as experimental materials to screen the candidate genes for the biosynthetic pathway of Cu Ⅱa by comparing the transcriptome sequencing results.The essential genes such as SE,OSC,CYP450 and acetyltransferase(AT)were screened and cloned.And the protein properties such as amino acids composition,domains,secondary structure and tertiary structure were analyzed by bioinformatics.The catalytic function of gene was studied using Escherichia coli(E.coli)and Saccharomyces cerevisiae(S.cerevisiae).The main research contents and results are as follows:1.Fifty-six functional genes related to Cu Ⅱa synthesis were screened from the local genome database using the Hemsleya transcriptome database,gene function annotation and comparison of homologous sequence.Ten of these genes were involved in the middle and downstream of the biosynthesis of Cu Ⅱa.HmSE1,HmSE2,and HmSE3 for SE genes;one cucurbitadienol synthase(CS)gene,HmCS;one HmCAS gene;one Hmβ-AS gene;and three CYP450 enzyme genes,HmCYP87,HmCYP81,and HmCYP705;one HmAT gene.The tuber c DNA was selected as the template based on the relative expression level in different tissues of Hemsleya macrosperma plants.Nine genes were successfully cloned except HmCS.2.Bioinformatics analysis showed that the three proteins encoded by HmSE1,HmSE2 and HmSE3 were hydrophobic proteins.HmSE1,HmCYP705 and HmAT encoded relatively stable proteins.The protein tertiary structure predicted showed that all proteins mainly had alpha helix and random coil.The results were consistent with the prediction results of protein secondary structure.Depending on the ratio of rare codons,E.coli BL21(DE3)was selected for HmSE1,HmAT protein prokaryotic expression or E.coli Transetta(DE3)was used for all genes.The proteins of HmCAS,Hmβ-AS and HmAT had no transmembrane structure and were located on the cell surface.The other six proteins had transmembrane domains.Phylogenetic tree results showed that the ten genes,including HmSE1 and HmSE2,respectively clustered with corresponding functional genes of other Cucurbitaceae plants.3.The prokaryotic expression vector pET-32a(+)-HmSE1 was constructed by the in-fusion cloning method.Then it was successfully expressed in E.coli Transetta(DE3)under the conditions of 0.5-1.0 m M Isopropyl β-D-thiogalactoside(IPTG)and cultured at 37℃ for 4hours.The weight of HmSE1 recombinant proteins with molecular was 76.74 k Da.The function of the HmSE1 protein was verified by enzymatic reaction in vitro.The GC-MS results showed that HmSE1 protein could catalyze squalene to 2,3-epoxysqualene.4.The eukaryotic expression vectors of pESC-TRP-HmXX,including HmSE1,HmSE2,HmSE3,HmCAS,Hmβ-AS,HmCYP87,HmAT genes,were constructed by the In-Fusion Cloning method.Then they were successfully expressed in S.cerevisiae WAT Ⅱ under the conditions of 2% galactose.Adding the substrates or using the 2,3-epoxysqualene of S.cerevisiae for fermentation.GC-MS or LC-MS detected the purified product.The results showed that the fermentation product of HmSE was the same as the 2,3-epoxysqualene produced by S.cerevisiae through its endogenous pathway,which would affect the product detection.Therefore,prokaryotic expression was selected to verify the products.HmCAS protein-catalyzed ergosterol synthesis increased the yield by about 2-fold,showing that the protein could catalyze 2,3-epoxysqualene to cycloartenol.And the cycloartenol would further generate ergosterol in S.cerevisiae.The new product catalyzed by Hmβ-AS protein was not consistent with the expectation,and it showed that the protein could not catalyze 2,3-epoxysqualene to β-amyrin in S.cerevisiae.The new product catalyzed by the HmCYP87 protein was challenging to be identified as 11-oxo-20β-hydroxy cucurbitadienol.Therefore,the system needed to be further scaled up,and NMR would determine the structure of the product after purification.The HmAT protein could catalyze cucurbitacin Ⅱb to Cu Ⅱa.In conclusion,based on the Hemsleya transcriptome database,this study screened,cloned and biogenic analyzed the nine genes of HmSE,HmOSC,the HmCYP450 hydroxylase and HmAT.And initially clarified the three key steps of squalene to 2,3-epoxysqualene,2,3-epoxysqualene cyclization and cucurbitacin Ⅱb to Cu Ⅱa through heterologous expressed.It will provide a basis for completely elucidating the Cu Ⅱa biosynthesis pathway.