Identification And Functional Analysis Of A C-28 Oxidase Involved In The Biosynthesis Of Tritepenoid Saponins From Ilex Asprella

ObjectiveTriterpenoid saponins(TSs)are a class of important secondary metabolites,which demonstrate various biological activities.However,the difficulties in their separation and purification as well as the complexity in chemical synthesis dramatically restrict the access of these components.Therefore,synthesis of TSs with engineered microorganisms could be an attractive alternative mode of production.Hex pubescens Hook.Et Arn.is a popular medicinal plant used in South China.The plant contains mainly ursane-type TS,which have been proved to be responsible for its pharmacological activities.Chemically,all the TSs of I.pubescens have a carboxyl group at C-28 site,implying the carboxylation at C-28 is the key reaction of TSs,biosynthesis.Therefore,the purpose of this study is to identify the key CYP gene catalyzing the formation of carboxyl group at C-28,laying an important foundation for the production of Ilex TSs in heterologous host organisms.Methods1.Screening of CYP candidate gene from I.pubescens transcriptomeTen well-characterized CYPs from other plant species involved in the oxidation of C-28 triterpenoids were used as subject to BLASTp the transcriptome of I.pubescens.The transcripts with sequence similarity over 40%were screened and further analyzed by using phylogenetie analysis.The transcript most closely-related to the characterized CYPs were selected as the candidate gene.2.Cloning of candidate gene and construction of expression plasmidsTotal RNA was extracted from the leaves of I.pubescens and cDNA was prepared by reverse transcription.Using cDNA as template,the candidate gene was cloned.The gene together with a triterpene cyclase gene IpAS1 identified previously were inserted into the two expression cassettes of yeast vector pESC-TRP by In-FusionnTM technology,respectively,to give the yeast expression construct.3.Heterologous expression and identification of secondary metabolitesUsing lithium acetate transfection method,the yeast expression construct was transformed into Saccharomyces cerevisiae WAT11,which can specifically express Arabidopsis cytochrome P450 reductase(CPR).The recombinant yeast was cultured in SC drop-out medium and induced with 2%galactose.Western Blot was used to evaluate the expression of recombinant protein.The expected metabolites,ursolic acid and oleanolic acid,were determined by GC-MS.4.Codon optimization and overexpression of tHMGR to regulate the metabolism of recombinant yeastAccording to the codon usage bias of S.cerevisiae,the sequence of candidate gene and IpAS1 were optimized and synthesized artificially.A novel yeast expression construct was generated and transferred to WAT11 accordingly.On the other hand,the gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR),one of the main rate-limiting enzymes involved in terpene biosynthesis,was transferred into the recombinant yeast both as free plasmid and insertion in the genome.The production of ursolic acid was compared with that of the recombinant yeast carrying only the native genes.Results1.CYP Candidate Gene from I.pubescens transcriptomeBased on the results of BLASTp and phylogenetic analysis,a transcript was screened,which contains an open reading frame of 1437 bp,encoding a protein of 478 aa(designated as IpAO1).IpAO1 showed the highest similarity with the multifunctional C-28 oxidase CYP716AL1,implying it might be involved in the C-28 position oxidation of triterpenoid saponins of I.pubescens.2.Functional identification of IpAO1The candidate gene IpAO1 was cloned and inserted into the yeast expression vector together with IpAS1,resulting in the plasmid pJL-AS1+AO1.Western blot analysis of the total protein extracted from the recombinant yeast WAT11/pJL-AS1+AO1 showed that IpAS1 and IpAO1 were successfully expressed after induction.The strain produced both oleanolic acid and ursolic acid,as compared to yeast strain expressing only IpAS1,which accumulated α-amyrin and β-amyrin.The results demonstrated that IpAO1 catalyzes the oxidation at the C-28 position of α-amyrin and β-amyrin to yield ursolic acid and oleanolic acid,respectively.3.Improvement of the titer of ursolic acid by codon optimization and overexpression of tHMGRTwo codon-optimized genes(opIpAS1 and opIpAO1)were synthesized and co-expressed in yeast WAT11.Compared to the recombinant yeast WATll/pJL-AS1+AO1,the strain with opIpAS1 and opIpAO1 accumulated more ursolic acid.The increase was estimated at 3.8-fold.Furthermore,overexpression of tHMGR gene also contributed to the production of ursolic acid.2.07-fold and 3.36-fold increase was observed for the recombinant yeast WATll/pJL-AS1+AO1/pJL-tHMGR and WATll-T/pJL-AS1+AO1,respectively.ConclusionIn this study,the CYP gene IpAO1 responsible for the oxidation of C-28 to form carboxyl group from I.pubescens was suceessfully cloned and functionally analyzed This is the first time that a CYP has been identified in Ilex genus.Furthermore,codon optimization of IpAO1 and overexpression of tHMGR clearly improved the ability of recombinant yeast to product ursolic acid,which may contribute to the study on heterologous biosynthesis of the biologically active Ilex TSs in yeast.