Identification Of IaAO2 And IaCPR2 Genes Involved In Triterpene Oxidation Modification From Ilex Asprella

ObjectiveIlex-asprella is a kind of traditional Chinese medicine commonly used in South China,the main components of the medicinal plant Ilex asprella are the ursane-type triterpenoids,of which the structural diversity is closely related to cytochrome P450 monooxygenases(CYPs),a class of key enzymes downstream of their biosynthetic pathways.Cytochrome P450 reductase(CPR)is an important component of cytochrome P450 system.Therefore,the aim of this study is to discover the cytochrome P450 monooxygenasegene gene(CYP)and cytochrome P450 reductase gene(CPR)involved in triterpenoid oxidation modification from the transcriptome data of Ilex asprella,clone and carry out functional studies,so as to lay a foundation for further production of ursane-type triterpenoids in Saccharomyces cerevisiae by metabolic engineering measures.Methods1.Screening of CYP and CPR genesUnigene or Contig,annotated as "cytochrome P450" and longer than 1200 bp,was retrieved from the transcriptome data of Ilex asprella.The amino acid sequence of the selected CYP gene was merged with the identified CYPs amino acid sequence(33 in total)involved in triterpene oxidation to construct phylogenetic tree.Unigene or Contig containing complete open reading frames were selected,and candidate genes were screened by combining phylogenetic tree and gene expression level.Unigene or Contig with full open reading frame annotated "cytochrome P450 reductase" was selected as candidate gene by retrieving data from transcriptome of Ilex asprella.2.Cloning of target genes and construction of recombinant expression plasmidsTotal RNA was extracted from tender leaves of Ilex asprella and retranscribed to obtain the cDNA.Specific primers were designed according to the transcriptome data.Target gene fragments were amplified by PCR using the cDNA as template.The target gene was linked to the zero background cloning vector and inserted into the expression vectors by ClonExpress Ⅱ One Step Cloning Kit to construct the recombinant expression plasmids.3.Expressing CYP gene in S.cerevisiae and identifying its functionThe recombinant plasmid carrying the target CYP gene was transformed into S.cerevisiae WAT11tfAX,which can synthesiz a-amyrin efficiently.The expression of CYP genes was induced by 2%galactose,and the expression of recombinant protein was analyzed by Western blot.The metabolites of recombinant yeast were detected by GC-MS to clarify the function of the CYP gene.4.Construct various S.cerevisiae strains and analyze the yield of ursolic acid in each recombinant yeastSeveral yeast recombinant expression plasmids were transformed into S.cerevisiae WAT11tfAX.Metabolites of recombinant yeasts were analyzed after 7 days induction and culture.The yield of ursolic acid was calculated and the functions of different genes were compared.5.Prokaryotic expression of the target geneThe recombinant expression plasmid was transformed into E.coli Transetta(DE3),and the recombinant protein was induced by 1 mM IPTG.Purify the target protein and analyze its enzymatic properties.Results1.Screening CYP and CPR genesOne CYP gene and two CPR genes possibly involved in triterpenoid oxidation were identified from the transcriptome data of Ilex asprella.They were named IaCPR1,IaCPR2 and IaAO2,respectively.2.Cloning of target gene and construction of recombinant expression plasmidIaAO2,IaCPR1 and IaCPR2 were cloned,recombinant yeast expression plasmids pESC-TRP-IaAO2,pESC-TRP-IaAO2-IaCPR1,pESC-TRP-IaAO2-IaCPR2,pESC-TRPtIaAO1 and recombinant E.coli expression plasmids pMAL-c5X-IaAO2,pET32a-tIaCPR1,pET32a-tIaCPR2 were constructed.3.Functional Identification of IaAO2 GeneThe recombinant plasmid pESC-TRP-IaAO2 was transformed into yeast strain WAT11tfAX to induce the expression of IaAO2 protein.Western blot analysis showed that the recombinant yeast could express IaAO2 protein of the expected size.The metabolites of recombinant yeast were analyzed by GC-MS.It was found that IaAO2 could oxidize aamyrin and β-amyrin to ursolic acid and oleanolic acid respectively,with triterpene C-28 site oxidation function.4.Analysis of oleanolic acid and ursolic acid production in recombinant yeastsThe metabolites of five recombinant yeast strains WAT11tfAX/pT-AO2,WAT11tfAX/pT-AO2-CPR1,WAT11tfAX/pT-AO2-CPR2,WAT11tfAX/pT-AO1 and WAT11tfAX/pT-tAO1 after 7 days of induction culture were analyzed.The results showed that the WAT11tfAX/pT-AO2 strain transformed with the recombinant expression plasmid carrying the IaAO2 gene accumulated 388.22 μg/L ursolic acid.When the gene IaCPRl was introduced,the content of ursolic acid in WAT11tfAX/pT-AO2-CPR1 strain did not change significantly.When the gene IaCPR2 was introduced,the content of ursolic acid in WAT11tfAX/pT-AO2-CPR2 strain increased to 964.73μg/L,indicating that the expression of IaCPR2 protein can improve the catalytic efficiency of P450 system in recombinant yeast.The WAT11tfAX/pT-AO1 strain constructed earlier by our research group can express the IaAO1 gene of Ilex asprella,which can accumulate 731.70 ug/L ursolic acid.The WAT11tfAX/pT-AO1 strain constructed earlier by our research group can express the IaAO1 gene,which can accumulate 731.70 ug/L ursolic acid,after truncating the Nterminus of IaAO1 protein,the tIaAO1 protein still has the function of oxidizing triterpenoid C-28,which can oxidize the substrates α-amyrin and β-amyrin to oleanolic acid and ursolic acid,respectively,but its catalytic efficiency is reduced.The accumulated ursolic acid content decreased to 421.16 μg/L.5.Prokaryotic expression of IaCPRl,IaCPR2 and IaAO2 genesRecombinant protein IaCPR1,IaCPR2 and IaAO2 were successfully expressed in E.coli Transetta(DE3)and their enzymatic properties were analyzed.ConclusionIn this study,a cytochrome P450 monooxygenase gene IaAO2 involved in triterpene C-28 oxidation and two cytochrome P450 reductase genes,IaCPRl and IaCPR2 of Ilex asprella were cloned and identified.IaAO2 can oxidize α-amyrin and β-amyrin to ursolic acid and oleanolic acid,respectively.Furthermore,it was found that the accumulation of ursolic acid in recombinant yeast increased significantly when IaAO2 and IaCPR2 genes were co-expressed,compared with the IaAO2 gene expressed alone,suggesting that IaCPR2 protein could improve the catalytic efficiency of heterologous P450 system in S.cerevisiae.