• Molecules and Cells
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• v.19 no.2
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• pp.294-299
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• 2005

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.

• Korean Journal of Agricultural Science
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• v.37 no.3
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• pp.367-371
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• 2010

Farnesyl diphosphate synthase (FPS) catalyzes the biosynthesis of farnesyl diphosphate, a precursor for many important terpenoid products. A cDNA encoding FPS was first isolated from Allium sativum (AsFPS) using rapid amplification of cDNA ends (RACE) PCR. The sequence of AsFPS contains an open reading frame encoding a protein of 341 amino acids with a predicted molecular mass of 39.61 kDa. Alignment of AsFPS deduced amino acid revealed high identities with other plants ranging from 79% to 85% and showed 2-high conserved aspartate-rich motifs known to be important for FPS activity. Furthermore, AsFPS expression was stronger in the green organs such as bulbils, scapes, leaves, stems, but weaker in bulbs and roots than on-green organs of A. sativum.

• Korean Journal of Medicinal Crop Science
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• v.21 no.1
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• pp.32-38
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• 2013

FPS (farnesyl diphosphate synthase) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. In order to investigate the effect of FPS on ginsenosides biosynthesis, we over-expressed FPS of Centella asiatica (CaFPS) in Panax giseng adventitious roots. PCR analysis showed the integrations of the CaFPS and hygromycin phosphotransferase genes and we ultimately selected three lines. The result of Southern blot analysis demonstrated the introduction of the CaFPS gene into genome of ginseng. In addition, the results of RT-PCR analysis revealed that CaFPS gene overexpression induced an accumulation of its transcription in the ginseng adventitious roots. To determine whether or not the overexpression of the CaFPS gene contributes to the downstream gene expression associated with triterpene biosynthesis, the level of mRNAs was analyzed by real-time PCR. The result showed that no differences were detected in any expression of all genes. To determine quantitatively the content of ginsenosides in transgenic ginseng adventitious roots, HPLC analysis was conducted. The content of total 7 ginsenosides was increased to 1.8, 1.4, and 1.7 times than that of the controls, respectively. This indicated that the overexpression of CaFPS in ginseng adventitious roots causes an increase in ginsenoside content, although down stream genes of FPS gene were suppressed by CaFPS overexpression.

• 한국약용작물학회:학술대회논문집
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• 2007.11a
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• pp.322.2-322.2
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• 2007

• Journal of Life Science
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• v.18 no.12
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• pp.1764-1770
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• 2008

The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ${\mu}g$/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ${\mu}g$/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.222-227
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• 2002

Squalene synthase catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene at the final branch point of the cholesterol biosynthetic pathway. Due to the unique position of this enzyme in the pathway, its inhibitors may have advantages as antihypercholesterolemic agents. Therefore, selective inhibitors of squalene synthase do not prevent the formation of the essential branch products of the isoprene pathway, such as dolichol, coenzyme-Q, and prenylated proteins, as might be expected for inhibitors of enzymes earlier in the pathway; for example, lovastatin and mevalotin. The current study reports that CJ90002, a pentagalloylglucose isolated from Paeonia moutan SIM (Paeoniaceae), which is an important Chinese crude drug used in many traditional prescriptions, was a potent inhibitor of rat microsomal squalene synthase, and also a potent inhibitor of cholesterol biosynthesis in vitro. In addition, the intraperitoneal and oral administration of CJ90002 had a significant lowering effect on plasma cholesterol levels in hamsters.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1216-1220
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• 2008

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The full-length recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the $Mg^{2+}$-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.759-765
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• 2013

The gene encoding squalene synthase (SQS) of the lipid-producing heterotrophic microalga Aurantiochytrium sp. KRS101 was cloned and characterized. The krsSQS gene is 1,551 bp in length and has two exons and one intron. The open reading frame of the gene is 1,164 bp in length, yielding a polypeptide of 387 predicted amino acid residues with a molecular mass of 42.7 kDa. The deduced krsSQS sequence shares at least four conserved regions known to be required for SQS enzymatic activity in other species. The protein, tagged with $His_6$, was expressed into soluble form in Escherichia coli. The purified protein catalyzed the conversion of farnesyl diphosphate to squalene in the presence of NADPH and $Mg^{2+}$. This is the first report on the characterization of an SQS from a Thraustochytrid microalga.

• Journal of Ginseng Research
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• v.34 no.1
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• pp.17-22
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• 2010

Sesquiterpenes are found naturally in plants and insects as defensive agents or pheromones. They are produced in the cytosolic acetate/mevalonate pathway for isoprenoid biosynthesis. The inducible sesquiterpene synthases (STS), which are responsible for the transformation of the precursor farnesyl diphosphate, appear to generate very few olefinic products that are converted to biologically active metabolites. In this study, we isolated the STS gene from Panax ginseng C.A. Meyer, designated PgSTS, and investigated the correlation between its expression and various abiotic stresses using real-time PCR. PgSTS cDNA was observed to be 1,883 nucleotides long with an open reading frame of 1,707 bp, encoding a protein of 568 amino acids. The molecular mass of the mature protein was determined to be 65.5 kDa, with a predicted isoelectric point of 5.98. A GenBank BlastX search revealed the deduced amino acid sequence of PgSTS to be homologous to STS from other plants, with the highest similarity to an STS from Lycopersicon hirsutum (55% identity, 51% similarity). Real-time PCR analysis showed that different abiotic stresses triggered significant induction of PgSTS expression at different time points.

• BMB Reports
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• v.40 no.5
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• pp.625-635
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• 2007

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semi-quantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.