Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis)

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.     

Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39 420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.     

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil,Anthonomus grandis

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full‐length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three‐dimensional structure of coleopteran FPPS was determined and compared to the X‐ray crystal structure of avian FPPS. The α‐helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. © 2009 Wiley Periodicals, Inc.     

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.     

Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.     

Cloning and characterization of decaprenyl diphosphate synthase from three different fungi

Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ₁₀ in addition to endogenous CoQ₈. Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombe dps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ₁₀, indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ₁₀ in other organisms.

     

卷叶贝母法尼基焦磷酸合酶基因的克隆及表达分析

为了探究卷叶贝母(Fritillaria cirrhosa)法尼基焦磷酸合酶基因(FcFPPS)是否参与甾类生物碱合成、萜类合成等代谢过程,该研究基于转录组测序结果,通过PCR技术克隆卷叶贝母FPPS基因(FcFPPS)开放阅读框(Open Reading Frame,ORF)序列,运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,并通过qRT-PCR检测FcFPPS基因在野生鳞茎和再生鳞茎(通过激素组合刺激获得的组织培养物)中的表达情况,以及利用煎煮法测定野生鳞茎和再生鳞茎的总生物碱含量。结果表明:获得了1 059bp的FcFPPS ORF片段,编码352个氨基酸,并与NCBI上公布的麝香百合、虎眼万年青、春兰等植物FPPS蛋白的相似性在85%以上;对FcFPPS蛋白的二级、三级结构预测发现FcFPPS蛋白主要由α螺旋构成;qRT-PCR与总生物碱含量测定结果显示FcFPPS基因的表达水平与总生物碱含量的变化趋势一致,都是再生鳞茎高于野生鳞茎。FcFPPS蛋白质特征区及同源性等生物信息学分析结合qRT-PCR的测定结果证明FcFPPS可能是一个有生物学功能的蛋白质,这为后续利用基因工程手段提高卷叶贝母中生物碱含量奠定了理论基础。     

Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.     

Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.     

Localization of farnesyl diphosphate synthase in chloroplasts.

The subcellular localization of plant farnesyl diphosphate synthase (FPPS) was examined. Immunocytochemical staining using anti-FPPS1 antibody followed by electron microscopy showed that FPPS1 was localized to chloroplasts of rice mesophyll cells. Subcellular fractions from wheat leaves were examined by immunoblot analysis. FPPS was detected in the chloroplast fraction in wheat, and was protected from proteolysis following trypsin treatment of chloroplasts. FPPS was also detected in the chloroplast fraction of a dicot plant, tobacco.     

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.     

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.