Screening And Functional Characterization Of The Genes Related To Gypenoside Biosynthesis

To date,Gynostemma is the only genus containing ginsenosides other than Panax genus.Gynostemma pentaphyllum(Thunb.)Makino,belonging to the Cucurbitaceae family,is a kind of perennial creeping herbaceous plant.G.pentaphyllum,also known as"Southern Ginseng" in China.Gypenosides(protopanaxadiol-type saponins)are the main active components of G.pentaphyllum,possessing various pharmacological activities,such as antitumor,hypoglycemic,hypolipidemic,cardiovascular and cerebrovascular protection and immunoprotection.In recent years,the ginsenoside biosynthesis in Panax species has been extensively studied,but the gypenoside biosythesis in G.pentaphyllum has remained poorly unclear until now.The study on the gypenoside biosythetic pathway is of great scientific significance to reveal the evolutionary mechanism of dammarane-type saponin biosynthetic pathways in different plants,and will further promote the industrial production of dammarane-type saponins based on synthetic biology.In order to obtain the sequences related to the biosythesis of gypenosides,we sequenced the transcriptomes from the roots,stems,and leaves of G.pentaphyllum with PacBio RSⅡ and Illumina NextSeq 500 sequenceing platforms.Following the PacBio standard analysis pipeline,66,046 polished consensus sequences were obtained,while Illumina data were assembled into 140,601 unigenes with Trinity software.Then,these output sequences from the two analytical routes were merged.After removing redundant data,a total of 140,157 final unigenes were generated with an average length of 750 bp.After functional annotation,five 2,3-oxidosqualene cyclase genes,145 cytochrome P450 genes,254 UDP-glycosyltransferase genes and 1362 Transcriptional factors were selected for the screening of genes involved in gypenoside biosynthesis.In general,the genes encoding enzymes with the same or similar catalytic activities are usually clustered together in the phylogenetic tree.In this work,we found that several genes were divided into the same families or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis.GpOSCl and GpOSC4 were phylogenetically grouped to the β-AS clade and CbQ clade,respectively;GpCYP716A1 and GpCYP716A2 were clustered into the same clade with the P450s involved in ginsenoside biosynthesis in P.ginseng,which belonged to the CYP716 family;13 GpUGTs and six GpUGTs were clustered into the same clade with UGTs for ginsenoside biosynthesis,belonging to the UGT71,UGT74,and UGT94 families,respectively;13 GpWRKYs and PqWRKY1 belonged to the Ⅱc subgroup;four GpbHLHs and PnbHLH1 classified into the Ⅳa subgroup.In general,the genes encoding enzymes in the same pathway are co-expressed.Therfore,co-expression analysis was used to further narrow down the range of the candidate genes.In this study,with the identified genes involved in upstream pathway as references,we analyzed the expression levels of the candidate genes in different tissues and after MeJA treatment using real-time PCR.According to the results of real-time PCR,Gp OSCl,GpCYP716A2 and GpUGT35 showed similar gene expression patterns to the upstream genes(SS,SE and HMGR),that was,increased sequentially in the roots,stems and leaves.Moreover,they were induced by MeJA,and reached the highest expression level at 18 h.Therefore,we speculated that GpOSC1,GpCYP716A2 and GpUGT35 were the leading candidates for gypenoside biosynthesis.In addition,based on the results of co-expression analysis,six GpWRKYs and GpbHLH68 might be involved in the regulation of gypenoside biosynthesis.To determine the function of the candidate genes,the ORFs of GpOSC1,GpOSC4 and GpCYP716A2 were inserted into the pYES2-URA or pESC-URA expression vector and expressed in Saccharomyces cerevisiae under the control of the regulatable promoter(GAL1),respectively.The yeast extracts were analyzed using GC-MS.Based on the results of GC analysis,the host harboring GpOSC1 gene had a unique peak at 30.68 min(product 1)and the host harboring GpOSC4 gene had a unique peak at 13.21 min(product 2),which were corresponded to the retention time of standard Dammarenediol-Ⅱ and Cucurbitadienol,respectively.The structures of all these products were explained by comparison of mass spectral characteristics with those of the authentic standards.This result suggested that GpOSC1 encodes a Dammarenediol-Ⅱ synthase,while GpOSC4 encodes a Cucurbitadienol synthase.GpCYP716A2 was found to have unknown function after yeast expression,and further research will be needed.The RNAi and over-expression transgenic hairy roots of GpOSC1 were constructed.To further confirm the function of GpOSC1 in the biosynthesis of gypenosides,the content of gypenosides will be detected in the transgenic hairy roots of the over-expression lines or RNAi lines.In general,some key amino acid residues play important roles in the activities of enyzmes.Using human lanosterol synthase(PDB ID:1W6J)as a template,the three-dimensional protein models of GpOSC1 and PgDSⅡ were generated with Modeller 9.18 and the substrate 2,3-oxidosqualene was docked with the model structures using Autodocking tools,respectively.The results showed that the substrate entered the active sites of target proteins mainly through hydrophobic interactions and van der Waals forces.The binding energy of GpOSC1 was lower than that of PgDSⅡ,indicating that the complex of GpOSC1 and the substrate was more stable.Based on Homology modeling and molecular docking,several amino acids were presumed as active-site residues for the 2,3-oxidosqualene cyclization.To preliminarily discover the effects of these amino acids,seven sites were selected to directionally modifiy GpOSCl by site-directed mutagenesis.GC-MS analysis was performed after heterologous expression in S.cerevisiae.The mutants of C486A,C564A,H479A and Y259H resulted in varible decreases in the activity of the enzyme.And the D485N,S412F and W418A mutants resulted in stable but inactive proteins.Here,we found several candidate genes related to the gypenoside biosynthesis,combining transcriptome sequencing with phylogenetic analysis and co-expression analysis of the gene families.GpOSC1 and GpOSC4 were successfully characterized via heterologous expression in S.cerevisiae.Protein modeling and site-directed mutagenesis led us to identify seven amino acids that are critical for the function of GpOSC1,which was also the first study to explore relationship between the structure and function of dammarenediol-Ⅱ synthase.In summary,our study will not only elucidate the biosynthetic pathway of gypenosides and provide useful components for the synthetic biology of gypenosides,but also lay a solid foundation for improving enzymes activity through directional modification.