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Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

ABSTRACT: Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

SUBMITTER: Wang J

PROVIDER: S-EPMC4284702 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

Wang Jianrong J Li Yangyuan Y Liu Danni D

International journal of molecular sciences 20141202 12

Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight o ...[more]

PMID: 25474088

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Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Project description:Farnesyl diphosphate synthase (FPS), a key enzyme of the terpene metabolic pathway, catalyzes the precursor of sesquiterpene compounds farnesyl diphosphate (FPP) synthesis, and plays an important role in regulating plant growth and development. Dryopteris fragrans is a medicinal plant rich terpenoids. In this study, the function of the gene was verified in vitro and in vivo, the promoter of the gene was amplified and its transcriptional activity was analyzed. In the present study, we report the molecular cloning and functional characterization of DfFPS1 and DfFPS2, two FPS genes from D. fragrans. We found that the two genes were evolutionarily conserved. Both DfFPS genes were highly expressed in the gametophyte and mature sporophyte leaves, and their expression levels increased in response to methyl jasmonate (MeJA) and high temperature. Both DfFPS proteins were localized in the cytoplasm and could catalyze FPP synthesis in vitro. We also found that the overexpression of DfFPS genes in tobacco plants promoted secondary metabolite accumulation but exhibited negligible effect on plant growth and development. However, the transgenic plants exhibited tolerance to high temperature and drought. The promoters of the two genes were amplified using fusion primer and nested integrated polymerase chain reaction (FPNI-PCR). The promoter sequences were truncated and their activity was examined using the β-glucuronidase (GUS) gene reporter system in tobacco leaves, and we found that both genes were expressed in the stomata. The transcriptional activity of the promoters was found to be similar to the expression pattern of the genes, and the transcriptional core regions of the two genes were mainly between -943 bp and -740 bp of proDfFPS1. Therefore, we present a preliminary study on the function and transcriptional activity of the FPS genes of D. fragrans and provide a basis for the regulation of terpene metabolism in D. fragrans. The results also provide a novel basis for the elucidation of terpene metabolic pathways in ferns.

Dataset Information

Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

Publications

Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

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