Cloning And Characterization Of Key Enzyme Genes In Saponin Biosynthesis Pathway From Conyza Blinii And Combinatorial Biosynthesis Of Conyzasaponin In Saccharomyces Cerevisiae

Conyza blinii H.Lév is a genuine herb from Sichuan and Yunnan province,which is a member of the plant family Compositae.It has effects of heat-clearing,detoxifying,relieving cough and relieving asthma,which is a good medicine for treatment of chronic bronchitis.The main pharmacological compounds of this plant are a class of oleanane-type pentacyclic triterpene glycosides,Conyzasaponins.Their low contents in natural plant restrict C.blinii development and utilization.With the development of biotechnology,synthesis secondary metabolites in microbes has shown great application prospect.Currently,the researches about C.blinii are only focused on its pharmacological efficacy,no studies on the synthesis of conyzasaponins.In this study,we establish the transcriptome library of C.blinii leaves.By screening the library,we obtain a large number of genes involved in conyzasaponins biosynthesis pathway.Including the key enzyme genes involved in the formation of precursor 2,3-oxidosqualene.The oxidosqualene cyclase（OSC）family genes that catalyze the synthesis of skeleton of triterpenoid saponins.The cytochrome P450（CYP450）family genes and UDP-glycosyltransferase（UGT）family genes that regulate saponin structural diversity.By studying these genes we initially elucidated the conyzasaponins biosynthesis metabolic flux.Through construction of conyzasaponin biosynthesis pathway in Saccharomyces cerevisiae,theβ-amyrin,oleanolic acid and 3-O-Glc-oleanolic acid are successfully produced,which provide theoretical basis and precursor materials for conyzasaponins biosynthesis in S.cerevisiae.The results of this study are as follows:1、Based on the Illumina Solexa Hi Seq 2500 sequencing system,42,903,527 read sequences are obtained from the leaves of Conyza blinii and successfully construct the transcriptome library.After assembly,80,930 Unigene sequences are obtained.Among them 45.27%are annotated in the Gen Bank database,30.33%are annotated in the protein family database and 32.36%are annotated in the Swiss-Prot database;According to the Gene Ontology database,these Unigenes are divided into three main categories cellular component,molecular function and biological process containing 52 subcategories;According to the eu Karyotic Orthologous Groups database,these Unigenes are divided into four main categories information storage and processing,cellular processes and signaling,metabolism and poorly characterized containing 25 subcategories.And by searching the Kyoto Encyclopedia of Genes and Genomes database,it is found that these Unigenes are annotated to 117 metabolic pathways.These pathways contain terpenoid backbone biosynthesis（ko00900）pathway,diterpenoid biosynthesis（ko00904）pathway,phenylpropanoid biosynthesis（ko00940）pathway,flavonoid biosynthesis（ko00941）pathway,flavone and flavonol biosynthesis（ko00944）pathway,N-Glycan biosynthesis（ko00510）pathway and other types of O-glycan biosynthesis（ko00514）pathway,which are related to the synthesis of the main compound conyzasaponin,the characteristic product blinin,flavonoids and polysaccharide,respectively.2、According to database annotation information select key enzyme genes involved in conyzasaponin biosynthetic pathway.As a result,three 3-hydroxy-3-methylglutaryl coenzyme A reductase（HMGR）genes,three farnesyl pyrophosphate synthase（FPPS）genes,one squalene synthase（SQS）gene,two squalene epoxidase（SQE）genes,ten oxidosqualene cyclase（OSC）genes,twenty-six cytochrome P450（CYP450）genes,seven cytochrome P450 reductase（CPR）genes and forty-seven UDP-glycosyltransferase（UGT）genes are discovered.After phylogenetic analysis one HMGR gene,one FPPS gene,one SQS gene,one SQE gene,one OSC（βAS）gene,two CYP450 genes,one CPR gene and eight UGT genes are selected as the key genes involved in conyzasaponin biosynthesis pathway.Further,eight most probable gene sequences are obtained by quantitative RT-PCR analysis after methyl jasmonate treatment.After PCR cloning,these genes are named Cb HMGR,Cb FPPS,Cb SQS,Cb SQE,CbβAS,Cb CYP716A261,Cb CPR and Cb UGT73C and then submitted to Gen Bank database.Bioinformatics tools are used to analyze characteristics of their nucleotide sequences and the deduced amino acid sequences.The results show that their characteristics are consistent with the characteristics of homologous genes.3、In order to detect the activity of the eight genes,the recombinant plasmids are constructed then expressed in Escherichia coli or Saccharomyces cerevisiae.The high performance liquid chromatography（HPLC）,gas chromatography-mass spectrometry（GC-MS）and spectrophotometry are used to detect the reaction products.The results are as following:（1）According to the GCMS results the specific peak produced by Cb HMGR at 8 min is mevalonic acid lactone,which indicated that Cb HMGR has the function of catalyzing the formation of mevalonate from HMG-Co A;（2）The detection of phosphorus by phosphorus molybdenum blue colorimetry showed that Cb FPPS can catalyze isopentenyl pyrophosphate and geranyl pyrophosphate to synthesize farnesyl pyrophosphate and pyrophosphate;（3）The catalytic reaction products of Cb SQS are detected by GCMS.The MS results show that the peak at 11.5 min in the experimental group is squalene,which indicated that Cb SQS can catalyze synthesis of squalene with farnesyl pyrophosphate as a substrate;（4）Detection of HPLC and comparison of 2,3-oxidosqualene standard suggest that Cb SQE has the function of catalyzing the formation of 2,3-oxidosqualene from squalene;（5）To verify the function of CbβAS,the yeast extracts are examined by GC-MS.The GC retention time show that at 19.5 min engineering strain and standardβ-amyrin appeared a specific peak.The results indicate that CbβAS can catalyze the production ofβ-amyrin;（6）The Cb CPR activity is detected by spectrophotometry.The results show that Cb CPR can transfer the electrons of NADPH to oxidized cytochrome C making it reduced with characteristic absorption peaks at 550 nm;（7）The microsomal protein catalyzed reaction product is detected by HPLC,which revealed that Cb CYP716A261 under aid of Cb CPR can catalyze the oxidation ofβ-amyrin at the C28 position,yielding oleanolic acid;（8）The Cb UGT73C catalytic products are detected by HPLC.The results show that the experimental group produce a new peak at 32.88 min,which suggests that Cb UGT73C might modify the C3position of oleanolic acid.4、We construct conyzasaponins biosynthesis pathway in Saccharomyces cerevisiae using endogenous 2,3-oxidosqualene as a substrate.The Cb CYP716A261 and Cb CPR genes are fused together by SOE PCR to obtain a new gene Cb CPR-linker-Cb CYP716A261 with a length of 3,573 bp.And then the fusion gene and CbβAS gene are constructed in p ESC-URA two multiple cloning sites and transformed into INVSc1.After galactose induction 18 h,q RT-PCR is used to monitor the gene expression levels to gain the engineering strain INVSc1-TM5.The results showed that TM5 yielded 35 mg/L oleanolic acid after induction by galactose for 60 h.Afterwards recombinant plasmid(Cb UGT73C-p ESC_His)is constructed and transferred into TM5.The SC-Ura-His plate and q RT-PCR are used to select engineering strain INVSc1-TM6.After galactose inductionthe yeast fermentation products are detected by HPLC.The results show that 3-O-Glc-oleanolic acid is successfully formed.The results reveal that the first step in biosynthesis of conyzasaponins is the cyclization of 2,3-oxidosqualene toβ-amyrin,a reaction that is catalyzed by CbβAS.Thenβ-amyrin is converted to oleanolic acid after direct oxidation by Cb CYP716A261.Finally oleanolic acid is converted to conyzasaponins after glycosylation by Cb UGT73C and other glycosyltransferases.