• Title/Summary/Keyword: isoprenoids

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Metabolic engineering for isoprenoids production in Escherichia coli

  • Kim, Seon-Won;Keasling, J.D.
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.70-73
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    • 2001
  • Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all isoprenoids. IPP in Escherichia coli is synthesized through the non-mevalonate pathway. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate(DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phosphate, catalyzed by DXP reductoismerase and encoded by dxr. To determine if one of more of the reactions in the non-mevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains ($DH5{\alpha}$, XL1-Blue, and JM101) that had been engineered to produce lycopene, a kind of isoprenoids. Lycopene production was improved significantly in strains transformed with the dex expression vectors. At arabinose concentrations between 0 and 1.33 mM, cells expressiong both dxs and from $P_{BAD}$ on a midium-copy plasmid produced 1.4 -2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cell expressing both dxs and dxr was lower than in cells expression dxs only. A comparison of the three E. coli strains trasfomed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.

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Triterpenoid Ginsenoside Biosynthesis in Panax ginseng C. A. Meyer (인삼에서의 트리터페노이드 진세노사이드의 생합성)

  • Kim, Yu-Jin;Lee, Ok-Ran;Yang, Deok-Chun
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2012.05a
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    • pp.20-20
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    • 2012
  • Isoprenoids represent the most diverse group of metabolites, which are functionally and structurally identified in plant organism to date. Ginsenosides, glycosylated triterpenes, are considered to be the major pharmaceutically active ingredient of ginseng. Its backbones, categorized as protopanaxadiol (PPD), protopanaxatriol (PPT), and oleanane saponin, are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene mediated with dammarenediol synthase or beta-amyrin synthase. The rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which is the first committed step enzyme catalyzes the cytoplasmic mevalonate (MVA) pathway for isoprenoid biosynthesis. DXP reductoisomerese (DXR), yields 2-C-methyl-D-erythritol 4-phosphate (MEP), is partly involved in isoprenoid biosynthesis via plastid. Squalene synthase and squalene epoxidase are involved right before the cyclization step. The triterpene backbone then undergoes various modifications, such as oxidation, substitution, and glycosylation. Here we will discuss general biosynthesis pathway for the production of ginsenoside and its modification based on their subcellular biological functions.

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Electrospray-Mass Spectrometric Analysis of Plasma Pyrophosphates Separated on a Multi-Modal Liquid Chromatographic Column

  • Lee, Su-Hyeon;Lee, Jeong-Ae;Lee, Won-Yong;Chung, Bong-Chul;Choi, Man-Ho
    • Mass Spectrometry Letters
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    • v.2 no.4
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    • pp.92-95
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    • 2011
  • Pyrophosphates are the key intermediates in the biosynthesis of isoprenoids, and their concentrations could reveal the benefits of statins in cardiovascular diseases. Quantitative analysis of five pyrophosphates, including isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative ionization mode. After dilution with methanol, samples were separated on a 3 ${\mu}m$ particle multi-modal $C_{18}$ column ($50{\times}2$ mm) and quantified within 10 min. The gradient elution consists of 10 mM ammonium bicarbonate and 0.5% triethylamine (TEA) in water and 0.1% TEA in 80% acetonitrile was used at the flow rate of 0.4 mL/min. Overall recoveries were 51.4-106.6%, while the limit of quantification was 0.05 ${\mu}g$/mL for GPP and FPP and 0.1 ${\mu}g$/mL for IPP, DMAPP, and GGPP. The precision (% CV) and accuracy (% bias) of the assay were 1.9-12.3% and 89.6-111.8%, respectively, in 0.05-10 ${\mu}g$/mL calibration ranges ($R^2$ > 0.993). The devised LC-MS/MS technique with the multi-modal $C_{18}$ column can be used to estimate the biological activity of pyrophosphates in plasma and may be applicable to cardiovascular events with cholesterol metabolism as well as the drug efficacy of statins.

Carotenoids Biosynthesis and Their Metabolic Engineering in Plants (식물에서 Carotenoid 생합성 경로와 대사공학적 응용)

  • Ha, Sun-Hwa;Kim, Jung-Bong;Park, Jong-Sug;Ryu, Tae-Hun;Kim, Kyung-Hwan;Hahn, Bum-Soo;Kim, Jong-Bum;Kim, Youg-Hwan
    • Journal of Plant Biotechnology
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    • v.30 no.1
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    • pp.81-95
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    • 2003
  • Carotenoids are synthesized from the plastidic glyceraldehyde-3-phosphate (GAP)/pyruvate pathway in isoprenoids biosynthetic system of plants. They play a crucial role in light harvesting, work as photoprotective agents in photosynthesis of nature, and are also responsible for the red, orange and yellow colors of fruits and flowers in plants. In addition to biological actions of carotenoids as antioxidants and natural pigments, they are essential components of human diet as a source of vitamin A. It has been also suggested that some kinds of carotenoids might provide protection against cancer and heart disease as human medicines. In this article, we review the commercial applications on the basis of biological functions of carotenoids, summarize the studies of genes involved in the carotenoid biosynthetic pathway, and introduce recent results achieved in metabolic engineering of carotenoids. This effort for understanding the carotenoids metabolism will make us to increase the total carotenoid contents of crop plants, direct the carotenoid biosynthetic machinery towards other useful carotenoids, and produce a new array of carotenoids by further metabolizing the new precursors that are created when one or two key enzymes in carotenoid biosynthetic pathway are exchanged through gene manipulation in the near future.

Expression Patterns of Genes Involved in Carotenoid Biosynthesis in Pepper

  • Ha, Sun-Hwa;Lee, Shin-Woo;Kim, Jong-Guk;Hwang, Young-Soo
    • Journal of Applied Biological Chemistry
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    • v.42 no.2
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    • pp.92-96
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    • 1999
  • To study the regulatory mechanism of isoprenoid (carotenoid) biosynthesis, we have compared the expression patterns of nine isoprenoid biosynthetic genes in Korean red pepper (Capsicum. annuum cv. NocKaung). The expression of geranylgeranyl pyrophosphate synthase gene was initially induced at early ripening stage (I1) and was rather slightly decreased during pepper fruit ripening. The ex-pression of phytoene synthase gene was strongly induced at semi-ripening stage (I2) and the phytoene desaturase transcript was maximally induced at the fully ripened stage (R). Our results suggest that genes encoding two 3-hydroxy-3-methylglutaryl-CoA reductase isozymes (HMGR1 and HMGR2) and farnesyl pyrophosphate synthase might be not so critical in pepper carotenoid biosynthesis but three genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase were induced in a sequential manner and coordinately regulated during the ripening of pepper fruit.

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Comparative Modeling Studies of 1-deoxy-D-xylulose 5-phosphate Synthase (MEP pathway) from Mycobacterium Tuberculosis

  • Kothandan, Gugan
    • Journal of Integrative Natural Science
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    • v.4 no.3
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    • pp.202-209
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    • 2011
  • Tuberculosis is a major health problem in humans because of its multidrug resistance and discovering new treatments for this disease is urgently required. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target. In this context, 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of M. tuberculosis has drawn attention. The MEP pathway begins with the condensation of glyceraldehyde 3-phosphate and pyruvate forming 1-deoxy-D-xylulose 5-phosphate (DXP) which is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS). As there is no X-ray structure was reported for this target, comparative modeling was used to generate the three dimensional structure. The structure was further validated by PROCHECK, VERIFY-3D, PROSA, ERRAT and WHATIF. Molecular docking studies was performed with the substrate (Thiamine pyrophosphate) and the reported inhibitor 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolol[1,5-a]pyrimidin-7-one) against the developed model to identify the crucial residues in the active site. This study may further be useful to provide structure based drug design.

디젤로 오염된 토양의 효과적인 Bioventing

  • 왕성환;오영진;문원재;박태주
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2002.04a
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    • pp.66-69
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    • 2002
  • In this work, cost effective venting is considered by comparing flow rates of 5$m\ell$/min, 10$m\ell$/min, and 20$m\ell$/min. Studies were performed on a soil artificially contaminated with diesel oil (the initial TPH(Total Petroleum Hydrocarbon) concentration of 7098mg/kg), and nutrient condition was C:N:P rate of 100:10:1. The soil has a sandy texture with pH of 6.8, 2.16 ~2.38% organic matter, a total porosity of 47~52% and field capacity 16.2~ 17.2%. The column experiments was made of glass column of 60cm length and 10cm I.D. at controlled temperature of 2$0^{\circ}C$($\pm$2.5$^{\circ}C$). The efficiency of continuous flow rate of 5, 10 and 20$m\ell$/min resulted in separately 61.3%, 58.1%, and 55% reduction of initial TPH concentration(7098mg/kg). Hydrocarbon utilizing microbial count and dehydrogenase activity in air flow of 5$m\ell$/min were higher than those of the others. The first order degradation rate of n-alkanes ranging from C10 to C28 was higher than that of pristane and phytane as isoprenoids. The $C_{17}$/pristane and $C_{18}$phytane ratios for monitoring the degree of biodegradation were useful only during the early stages of oil degradation. Degradation contributed from about 89% to 93% of TPH removal. Volatilization loss of diesel oil in contaminated soil was about 7% to 11%, which was significantly small compared to degradation.n.

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Heterogeneity of Human γδ T Cells and Their Role in Cancer Immunity

  • Hye Won Lee;Yun Shin Chung;Tae Jin Kim
    • IMMUNE NETWORK
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    • v.20 no.1
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    • pp.5.1-5.15
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    • 2020
  • The γδ T cells are unconventional lymphocytes that function in both innate and adaptive immune responses against various intracellular and infectious stresses. The γδ T cells can be exploited as cancer-killing effector cells since γδ TCRs recognize MHC-like molecules and growth factor receptors that are upregulated in cancer cells, and γδ T cells can differentiate into cytotoxic effector cells. However, γδ T cells may also promote tumor progression by secreting IL-17 or other cytokines. Therefore, it is essential to understand how the differentiation and homeostasis of γδ T cells are regulated and whether distinct γδ T cell subsets have different functions. Human γδ T cells are classified into Vδ2 and non-Vδ2 γδ T cells. The majority of Vδ2 γδ T cells are Vγ9δ2 T cells that recognize pyrophosphorylated isoprenoids generated by the dysregulated mevalonate pathway. In contrast, Vδ1 T cells expand from initially diverse TCR repertoire in patients with infectious diseases and cancers. The ligands of Vδ1 T cells are diverse and include the growth factor receptors such as endothelial protein C receptor. Both Vδ1 and Vδ2 γδ T cells are implicated to have immunotherapeutic potentials for cancers, but the detailed elucidation of the distinct characteristics of 2 populations will be required to enhance the immunotherapeutic potential of γδ T cells. Here, we summarize recent progress regarding cancer immunology of human γδ T cells, including their development, heterogeneity, and plasticity, the putative mechanisms underlying ligand recognition and activation, and their dual effects on tumor progression in the tumor microenvironment.

Statins and Their Effects on Embryonic Stem Cells (스타틴 그리고 배아줄기세포에서의 작용)

  • Lee, Mi-Hee;Han, Yong-Mahn;Cho, Yee-Sook
    • Development and Reproduction
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    • v.11 no.2
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    • pp.59-66
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    • 2007
  • Understanding molecular mechanisms that control embryonic stem cell (ESC) self-renewal and differentiation is important for the development of ESC-based therapies. Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), potently reduce cholesterol level. As well as inhibiting cholesterol synthesis, statins inhibit other intermediates in the mevalonate pathway such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), major substrates for protein isoprenylation. Studies showed that pleiotropic effects of statins beyond cholesterol lowering property arise from inhibition of protein isoprenylation that is involved in various cellular functions including proliferation and differentiation. It has been determined that statins have inhibitory effect on ESC self-renewal and stimulatory effect on ESC differentiation into adipogenic/osteogenic lineages. Importantly, statins mediate downregulation of ESC self-renewal by inhibiting RhoA-dependent signaling, independently of their choresterol-lowering properties. Understanding statin's actions on ESCs may provide important insights into the molecular mechanisms that regulate self-renewal or differentiation of ESCs.

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