Functional Analysis Of CsF3H And CsDFR And Construction Of Metabolicaily Engineered E.coli Producing Catechin

Catechins are important plant secondary metabolites,which have beneficial effects on human health.In plants,catechins and other flavonoids compounds are synthesized through phenylpropanoid pathway and flavonoids pathway using phenylpropionic acid as substrate.In the flavonoids pathway,F3 H can catalyze flavanones into dihydroflavonols,while DFR transfer dihydroflavonols into leucoanthocyanins,and then are transferred into catechins by leucoanthocyanins reductase(LAR).In this study,F3H?DFR1 and DFR2 genes were cloned from tea leaves and bioinformatically analyzed.Real-time fluorescent quantitative was used to analyze the different expressions of the three genes.Then we used prokaryotic expressions to obtain target recombinant proteins,and detected the enzyme activity.Meanwhile,we used prokaryotic expression technology to construct metabolic engineering bacteria for high-level catechin productions.The main results are as follows:1.Cloning and functional analysis of flavanone 3-hydroxylase geneIn our study,we amplified the ORF of CsF3 H by end-to-end PCR from the leaves of tea,which contained 1107 bp and encoded a protein with 368 amino acids.Real-time fluorescent quantitative was used to analyze the different expressions of CsF3 H gene in different organs.The result showed that CsF3 H had the highest expression level in the forth leaf.Then we used prokaryotic expression to express F3 H protein,and detected the recombinant enzyme activity in vitro by HPLC.The results indicated that recombinant protein had the F3 H enzyme activity and transfered naringenin(N)and eriodictyol(E)respectively to dihydrokaempfero(DHK)and dihydroquercetin(DHQ),while the protein showed higher activity when eriodictyol was used as substrate.2.Cloning and functional analysis of two dihydroflavonol 4-reductase genesThe ORF of CsDFR1 and CsDFR2 genes were obtained by end-to-end PCR.CsDFR1 contained 1044 bp and encoded a protein with 347 amino acids,while CsDFR2 contained 1035 bp and encoded a protein with 344 amino acids.qRT-PCR was used to analyze the relative expressions of CsDFR1 and CsDFR2 in different organs and treatment conditions.The results showed that CsDFR1 was expressed higher in bud and 3rd leaf,much lower in stem and root.While CsDFR2 were greatly expressed in 2nd,3rd and root,but lowest in stem.The responses to inducing conditions between CsDFR1 and CsDFR2 were different.CsDFR2 was vastly regulated in drought and UV treatments while CsDFR1 was clearly decreased to shading.The two genes were cloned into the expression vector sumo for prokaryotic expression,then transferred into BL21 induced by IPTG to express target proteins.The recombinant enzyme activity in vitro was detected by HPLC.The results indicated that recombinant proteins showed the DFR activity,and catalyzed dihydroflavonols into leucoanthocyanins.3.Engineering central flavonoid pathway for high-level catechin productions in Escherichia coliTwo groups of recombinant plasmids were constructed,pETDuet-LAR and pRSFDuet-F3H-DFR1,pETDuet-F3 H and pRSFDuet-LAR-DFR1.The two groups were transfered into BL21 respectively to conform recombinant E.coli FD-L(containing pETDuet-LAR and pRSFDuet-F3H-DFR1)and F-DL(containing pETDuet-F3 H and pRSFDuet-LAR-DFR1).Naringenin and eriodictyol were used as substrates to obtain catechins.The result showed that afzelechin was synthesized when using naringenin as substrate,and catechin was produced when using eriodictyol as substrate.We could reach a final catechin concentration up to 61.10 mg/L.Besides,E.coli FD-L was a bit more efficient than F-DL whichever the substrate was.