DOCSLIB.ORG

Investigation of the Methylerythritol 4-Phosphate Pathway for Microbial

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Investigation of the Methylerythritol 4-Phosphate Pathway for Microbial Volke et al. Microb Cell Fact (2019) 18:192 https://doi.org/10.1186/s12934-019-1235-5 Microbial Cell Factories RESEARCH Open Access Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis Daniel Christoph Volke1,2, Johann Rohwer3, Rainer Fischer1,4 and Stefan Jennewein1* Abstract Background: Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpe- noids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. Results: We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/ dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and iso- pentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a fux control coefcient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway fux) in the isoprene-pro- ducing strain and therefore exerted signifcant control over the fux though the MEP pathway. At higher dxs expres- sion levels, the intracellular concentration of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the fux towards isoprene. The higher intracellular concentrations of MEcPP led to the efux of this intermediate into the growth medium. Discussion: These fndings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineer- ing of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids. Keywords: MEP pathway, Metabolic control analysis, Recombineering, Isoprene, E. coli Highlights • Metabolomic and proteomic analysis of the MEP pathway. • Metabolic control analysis of the MEP pathway in E. • Reduction of MEcPP to HMBPP is a limiting step coli. for fux through the MEP pathway and promotes the • Dxs has high fux and concentration control over efux of MEcPP at higher fux rates. MEP pathway intermediates. Background *Correspondence: [email protected] Microbes can synthesize a broad spectrum of valuable 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany compounds and precursors, providing an inexpensive Full list of author information is available at the end of the article and sustainable source of industrially-relevant chemicals © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Volke et al. Microb Cell Fact (2019) 18:192 Page 2 of 15 including terpenoids. More than 60,000 terpenoids purifcation, makes isoprene a promising product for fer- have been described [1], ranging from pharmacologi- mentation process [9]. Te MCA framework was applied cally active molecules such as paclitaxel and artemisinin, to the concentrations of MEP pathway metabolites and which are used for the treatment of cancer and malaria, the fux towards isoprene according to diferent expres- respectively [2, 3], to promising biofuels such as sion levels of the dxs gene. To our knowledge, this is the farnesene [4]. All terpenoids originate from the iso- frst time that this approach was combined with recom- meric precursors dimethylallyl pyrophosphate (DMAPP) bineering, quantitative proteomics and metabolomics. and isopentenyl pyrophosphate (IPP), both of which are Tis combination yielded a quantitative description of produced via one of two natural metabolic routes: the the control of Dxs on the fux through and metabolite 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway concentrations in the MEP pathway, which will guide fur- and the mevalonate (MVA) pathway [5]. ther engineering eforts. Te MVA pathway has been engineered to produce large quantities of terpenoids [6, 7] but attempts to engi- Materials and methods neer the MEP pathway have been less successful [8, 9]. In Bacterial strains and culture conditions part, this refects our incomplete knowledge of the reg- Standard cloning and metabolic engineering were carried ulation and control of the MEP pathway, despite recent out using E. coli strains 10β and BL21 (DE3), respectively reports that have revealed a number of regulatory mech- (both supplied by New England Biolabs, Ipswich, MA, anisms [10–14]. Even so, the MEP pathway is a promis- USA). For general cloning, the bacteria were cultivated ing target for metabolic engineering because of its higher in lysogenic broth (LB) medium, whereas for all other theoretical yield (~ 20% more than the MVA pathway experiments the bacteria were cultivated in M9 medium under aerobic conditions in Escherichia coli) and its bal- supplemented with 0.5% (w/v) glucose and appropri- anced use of reducing equivalents [15, 16]. Attempts to ate antibiotics (50 μg mL−1 kanamycin, 100 μg mL−1 engineer the MEP pathway have thus far included rand- ampicillin and/or 25 μg mL−1 chloramphenicol) at 37 °C omization approaches, the combinatorial expression of [24]. Strains carrying the plasmids pSIM5 or pSIM6 [25] a subset of MEP pathway genes [17–20] and the opti- were grown at 30 °C and cured of the plasmids at 37 °C. mization of precursor supply [21]. Eforts to circumvent Cultures were grown in bafed Erlenmeyer fasks flled regulation by expressing the complete MEP pathway in to one-ffth of their nominal volume and agitated at heterologous hosts such as the yeast Saccharomyces cer- 180 rpm. Cell growth in liquid medium was monitored evisiae have not been successful [22]. Several studies have by spectrophotometry to determine the optical density at investigated the rate-limiting steps in the MEP pathway, 600 nm (OD600). but have failed to deliver a consistent picture [17, 18, 20]. Others have found indications for the intrinsic regulation General cloning and amplifcation of the MEP pathway at the steps catalyzed by 1-deoxy- General cloning procedures and plasmid purifcation d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy- were carried out according to standard laboratory prac- d-xylulose-5-phosphate reductoisomerase (Dxr) [10], tice [24]. Herculase II Fusion DNA Polymerase (Agilent 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase Technologies, Santa Clara, CA, USA) was used for the (IspD) [11] or 2-C-methyl-d-erythritol-2,4-cyclopyroph- amplifcation of DNA fragments by the polymerase chain osphate synthase (IspF) [12]. Te MEP pathway interme- reaction (PCR) according to the manufacturer’s instruc- diate 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate tions. All plasmids and altered genomic regions were (MEcPP) is found at higher concentrations in the extra- verifed by Sanger sequencing carried out by Eurofns cellular milieu in E. coli overexpressing MEP pathway GmbH (Ebersberg, Germany). genes [14]. However, the behavior of the MEP pathway has not been described in a precise and quantitative Construction of plasmids for the production of isoprene manner either in wild-type or engineered microbes. Te ispS gene from Populus alba was codon optimized We therefore set out to establish a quantitative descrip- for E. coli (Additional fle 1: Table S1) (Termo Fisher Sci- tion of the MEP pathway for terpenoid production using entifc, Waltham, MA, USA) and the gene was amplifed metabolic control analysis (MCA), a form of sensitivity using forward primer 5′-AAT AAT TTT GTT TAA CTT analysis that quantifes the efect of small changes in a TAA TAA GGA GAT ATA CCA TGG AAG CTC GTC given parameter (such as enzyme activity) on overall sys- GTT CTG C-3′ and reverse primer 5′-TTA GCG TTC tem characteristics such as metabolite concentration and AAA TGG CAG TAG CAA GCT TGT CGA CCA CGT fux [23]. Here we used the MEP pathway for the produc- TCG AAC GGC AGG ATC-3′ (start codon in bold). Vec- tion of isoprene. Besides having broad industrial appli- tor pCOLA was amplifed using forward primer 5′-GGT cations, its low boiling point, and therefore its ease of ATA TCT CCT TAT TAA AGT TAA ACA-3′ and reverse Volke et al. Microb Cell Fact (2019) 18:192 Page 3 of 15 primer 5′-GGT CGA CAA GCT TGC GGC CG-3′. GCC AAC GCG CTT CAA TG-3′ and reverse primer Te products were joined by Gibson assembly yielding 5′-TCG TGG TGA AGC AGA ACA AG-3′ for the dxr pCOLA::IspS, which was then amplifed using forward expression library. Te amplicons were sequenced using primer 5′-GGC CGC ATA ATG CTT AAG TCG-3′ and the same primers. reverse primer 5′-GCA AGC TTG TC GAC CTT AGC- 3′. Te isopentenyl diphosphate isomerase (idi) gene was Metabolite quantifcation amplifed from the genome of E. coli strain BL21 using A 10-mL volume of M9 medium supplemented with forward primer 5′-TAC TGC CAT TTG AAC GCT AAG 0.5% (w/v) glucose was inoculated with 100 µL of over- GTC GAC AAG CTT GCA AGG AGA TAT ACC ATG night culture in 200-mL Erlenmeyer fasks.
Load more

Recommended publications
  • WO 2013/180584 Al 5 December 2013 (05.12.2013) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/180584 Al 5 December 2013 (05.12.2013) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 1/21 (2006.01) C12N 15/74 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 15/52 (2006.01) C12P 5/02 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C12N 15/63 (2006.01) HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/NZ20 13/000095 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, (22) International Filing Date: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 4 June 2013 (04.06.2013) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 61/654,412 1 June 2012 (01 .06.2012) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (71) Applicant: LANZATECH NEW ZEALAND LIMITED MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, [NZ/NZ]; 24 Balfour Road, Parnell, Auckland, 1052 (NZ).
    [Show full text]
  • Summaries of FY 2001 Activities Energy Biosciences
    Summaries of FY 2001 Activities Energy Biosciences August 2002 ABSTRACTS OF PROJECTS SUPPORTED IN FY 2001 (NOTE: Dollar amounts are for a twelve-month period using FY 2001 funds unless otherwise stated) 1. U.S. Department of Agriculture Urbana, IL 61801 Biochemical and molecular analysis of a new control pathway in assimilate partitioning Daniel R. Bush, USDA-ARS and Department of Plant Biology, University of Illinois at Urbana- Champaign $72,666 (21 months) Plant leaves capture light energy from the sun and transform that energy into a useful form in the process called photosynthesis. The primary product of photosynthesis is sucrose. Generally, 50 to 80% of the sucrose synthesized is transported from the leaf to supply organic nutrients to many of the edible parts of the plant such as fruits, grains, and tubers. This resource allocation process is called assimilate partitioning and alterations in this system are known to significantly affect crop productivity. We recently discovered that sucrose plays a second vital role in assimilate partitioning by acting as a signal molecule that regulates the activity and gene expression of the proton-sucrose symporter that mediates long-distance sucrose transport. Research this year showed that symporter protein and transcripts turn-over with half-lives of about 2 hr and, therefore, sucrose transport activity and phloem loading are directly proportional to symporter transcription. Moreover, we showed that sucrose is a transcriptional regulator of symporter expression. We concluded from those results that sucrose-mediated transcriptional regulation of the sucrose symporter plays a key role in coordinating resource allocation in plants. 2. U.
    [Show full text]
  • S-Abscisic Acid
    CLH REPORT FOR[S-(Z,E)]-5-(1-HYDROXY-2,6,6-TRIMETHYL-4-OXOCYCLOHEX-2-EN- 1-YL)-3-METHYLPENTA-2,4-DIENOIC ACID; S-ABSCISIC ACID CLH report Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2 International Chemical Identification: [S-(Z,E)]-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2- en-1-yl)-3-methylpenta-2,4-dienoic acid; S-abscisic acid EC Number: 244-319-5 CAS Number: 21293-29-8 Index Number: - Contact details for dossier submitter: Bureau REACH National Institute for Public Health and the Environment (RIVM) The Netherlands [email protected] Version number: 1 Date: August 2018 Note on confidential information Please be aware that this report is intended to be made publicly available. Therefore it should not contain any confidential information. Such information should be provided in a separate confidential Annex to this report, clearly marked as such. [04.01-MF-003.01] CLH REPORT FOR[S-(Z,E)]-5-(1-HYDROXY-2,6,6-TRIMETHYL-4-OXOCYCLOHEX-2-EN- 1-YL)-3-METHYLPENTA-2,4-DIENOIC ACID; S-ABSCISIC ACID CONTENTS 1 IDENTITY OF THE SUBSTANCE........................................................................................................................1 1.1 NAME AND OTHER IDENTIFIERS OF THE SUBSTANCE...............................................................................................1 1.2 COMPOSITION OF THE SUBSTANCE..........................................................................................................................1 2 PROPOSED HARMONISED
    [Show full text]
  • Relationship to Atherosclerosis
    AN ABSTRACT OF THE THESIS OF Marilyn L. Walsh for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on May 3..2001. Title: Protocols. Pathways. Peptides and Redacted for Privacy Wilbert Gamble The vascular system transports components essential to the survival of the individual and acts as a bamer to substances that may injure the organism. Atherosclerosis is a dynamic, lesion producing disease of the arterial system that compromises the functioning of the organ by occlusive and thrombogenic processes. This investigation was undertaken to elucidate some of the normal biochemical processes related to the development of atherosclerosis. A significant part of the investigation was directed toward developing and combining methods and protocols to obtain the data in a concerted manner. A postmitochondnal supernatant of bovine aorta, usingmevalonate-2-14C as the substrate, was employed in the investigation. Methods included paper, thin layer, and silica gel chromatography; gel filtration, high performance liquid chromatography (HPLC), and mass spectrometry. This current research demonstrated direct incorporation of mevalonate-2- 14Cinto the trans-methyiglutaconic shunt intermediates. The aorta also contains alcohol dehydrogenase activity, which converts dimethylallyl alcohol and isopentenol to dimethylacrylic acid, a constituent of the trans-methylgiutaconate Small, radioactive peptides, named Nketewa as a group, were biosynthesized using mevalonate-2-'4C as the substrate. They were shown to pass through a 1000 D membrane. Acid hydrolysis and dabsyl-HPLC analysis defined the composition of the Nketewa peptides. One such peptide, Nketewa 1, had a molecular weight of 1038 and a sequence of his-gly-val-cys-phe-ala-ser-met (HGVCFASM), with afarnesyl group linked via thioether linkage to the cysteine residue.
    [Show full text]
  • Plasma Mevalonate As a Measure of Cholesterol Synthesis in Man
    Plasma mevalonate as a measure of cholesterol synthesis in man. T S Parker, … , J Chen, P J De Schepper J Clin Invest. 1984;74(3):795-804. https://doi.org/10.1172/JCI111495. Research Article Measurement of mevalonic acid (MVA) concentrations in plasma or 24-h urine samples is shown to be useful in studies of the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Plasma MVA concentrations, measured either at 7-9 a.m. after an overnight fast, or throughout the 24-h cycle, were compared with cholesterol synthesis rates that were measured by the sterol balance method: plasma MVA concentrations were directly related to the rate of whole body cholesterol synthesis (r = 0.972; p less than 0.001; n = 18) over a tenfold range of cholesterol synthesis rates. Moreover, hourly examination of MVA concentrations throughout the day demonstrated that interventions such as fasting or cholesterol feeding cause suppression of the postmidnight diurnal rise in plasma MVA concentrations, with little change in the base-line of the rhythm. Thus, the daily rise and fall of plasma MVA appears to reflect changes in tissues and organs, such as the liver and intestine, that are known to be most sensitive to regulation by fasting or by dietary cholesterol. The hypothesis that short-term regulation of HMG-CoA reductase in tissues is quickly reflected by corresponding variations in plasma MVA was tested by using a specific inhibitor of HMG-CoA reductase, mevinolin, to block MVA synthesis. Mevinolin caused a dose-dependent lowering of plasma MVA after a single dose; and in patients who […] Find the latest version: https://jci.me/111495/pdf Plasma Mevalonate as a Measure of Cholesterol Synthesis in Man Thomas S.
    [Show full text]
  • Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam
    Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam El Khawand A thesis submitted to the University of East Anglia in fulfillment of the requirements for the degree of Doctor of Philosophy School of Environmental Sciences November 2014 ©This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derive there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract Isoprene is a climate active gas emitted from natural and anthropogenic sources in quantities equivalent to the global methane flux to the atmosphere. 90 % of the emitted isoprene is produced enzymatically in the chloroplast of terrestrial plants from dimethylallyl pyrophosphate via the methylerythritol pathway. The main role of isoprene emission by plants is to reduce the damage caused by heat stress through stabilizing cellular membranes. Isoprene emission from microbes, animals, and humans has also been reported, albeit less understood than isoprene emission from plants. Despite large emissions, isoprene is present at low concentrations in the atmosphere due to its rapid reactions with other atmospheric components, such as hydroxyl radicals. Isoprene can extend the lifetime of potent greenhouse gases, influence the tropospheric concentrations of ozone, and induce the formation of secondary organic aerosols. While substantial knowledge exists about isoprene production and atmospheric chemistry, our knowledge of isoprene sinks is limited. Soils consume isoprene at a high rate and contain numerous isoprene-utilizing bacteria. However, Rhodococcus sp. AD45 is the only terrestrial isoprene-degrading bacterium characterized in any detail.
    [Show full text]
  • Medically Useful Plant Terpenoids: Biosynthesis, Occurrence, and Mechanism of Action
    molecules Review Medically Useful Plant Terpenoids: Biosynthesis, Occurrence, and Mechanism of Action Matthew E. Bergman 1 , Benjamin Davis 1 and Michael A. Phillips 1,2,* 1 Department of Cellular and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada; [email protected] (M.E.B.); [email protected] (B.D.) 2 Department of Biology, University of Toronto–Mississauga, Mississauga, ON L5L 1C6, Canada * Correspondence: [email protected]; Tel.: +1-905-569-4848 Academic Editors: Ewa Swiezewska, Liliana Surmacz and Bernhard Loll Received: 3 October 2019; Accepted: 30 October 2019; Published: 1 November 2019 Abstract: Specialized plant terpenoids have found fortuitous uses in medicine due to their evolutionary and biochemical selection for biological activity in animals. However, these highly functionalized natural products are produced through complex biosynthetic pathways for which we have a complete understanding in only a few cases. Here we review some of the most effective and promising plant terpenoids that are currently used in medicine and medical research and provide updates on their biosynthesis, natural occurrence, and mechanism of action in the body. This includes pharmacologically useful plastidic terpenoids such as p-menthane monoterpenoids, cannabinoids, paclitaxel (taxol®), and ingenol mebutate which are derived from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, as well as cytosolic terpenoids such as thapsigargin and artemisinin produced through the mevalonate (MVA) pathway. We further provide a review of the MEP and MVA precursor pathways which supply the carbon skeletons for the downstream transformations yielding these medically significant natural products. Keywords: isoprenoids; plant natural products; terpenoid biosynthesis; medicinal plants; terpene synthases; cytochrome P450s 1.
    [Show full text]
  • Metabolic Engineering of Escherichia Coli for Natural Product Biosynthesis
    Trends in Biotechnology Special Issue: Metabolic Engineering Review Metabolic Engineering of Escherichia coli for Natural Product Biosynthesis Dongsoo Yang,1,4 Seon Young Park,1,4 Yae Seul Park,1 Hyunmin Eun,1 and Sang Yup Lee1,2,3,∗ Natural products are widely employed in our daily lives as food additives, Highlights pharmaceuticals, nutraceuticals, and cosmetic ingredients, among others. E. coli has emerged as a prominent host However, their supply has often been limited because of low-yield extraction for natural product biosynthesis. from natural resources such as plants. To overcome this problem, metabolically Escherichia coli Improved enzymes with higher activity, engineered has emerged as a cell factory for natural product altered substrate specificity, and product biosynthesis because of many advantages including the availability of well- selectivity can be obtained by structure- established tools and strategies for metabolic engineering and high cell density based or computer simulation-based culture, in addition to its high growth rate. We review state-of-the-art metabolic protein engineering. E. coli engineering strategies for enhanced production of natural products in , Balancing the expression levels of genes together with representative examples. Future challenges and prospects of or pathway modules is effective in natural product biosynthesis by engineered E. coli are also discussed. increasing the metabolic flux towards target compounds. E. coli as a Cell Factory for Natural Product Biosynthesis System-wide analysis of metabolic Natural products have been widely used in food and medicine in human history. Many of these networks, omics analysis, adaptive natural products have been developed as pharmaceuticals or employed as structural backbones laboratory evolution, and biosensor- based screening can further increase for the development of new drugs [1], and also as food and cosmetic ingredients.
    [Show full text]
  • Isoprene Synthase and Polynucleotide Encoding Same, and Method for Producing Isoprene Monomer
    (19) TZZ Z_T (11) EP 2 857 509 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 08.04.2015 Bulletin 2015/15 C12N 15/09 (2006.01) C08F 36/08 (2006.01) C12N 1/21 (2006.01) C12N 9/88 (2006.01) (2006.01) (21) Application number: 13796298.1 C12P 5/02 (22) Date of filing: 12.03.2013 (86) International application number: PCT/JP2013/056866 (87) International publication number: WO 2013/179722 (05.12.2013 Gazette 2013/49) (84) Designated Contracting States: • FUKUSHIMA, Yasuo AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Kodaira-shi GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Tokyo 187-8531 (JP) PL PT RO RS SE SI SK SM TR • YOKOYAMA, Keiichi Designated Extension States: Kawasaki-shi BA ME Kanagawa 210-8681 (JP) • NISHIO, Yosuke (30) Priority: 30.05.2012 JP 2012123724 Kawasaki-shi 30.05.2012 JP 2012123728 Kanagawa 210-8681 (JP) • TAJIMA, Yoshinori (71) Applicants: Kawasaki-shi • Bridgestone Corporation Kanagawa 210-8681 (JP) Tokyo 104-8340 (JP) • MIHARA, Yoko • Ajinomoto Co., Inc. Kawasaki-shi Tokyo 104-8315 (JP) Kanagawa 210-8681 (JP) • NAKATA, Kunio (72) Inventors: Kawasaki-shi • HAYASHI, Yasuyuki Kanagawa 210-8681 (JP) Kodaira-shi Tokyo 187-8531 (JP) (74) Representative: Grünecker Patent- und • HARADA, Minako Rechtsanwälte Kodaira-shi PartG mbB Tokyo 187-8531 (JP) Anwaltssozietät • TAKAOKA, Saaya Leopoldstrasse 4 Kodaira-shi 80802 München (DE) Tokyo 187-8531 (JP) (54) ISOPRENE SYNTHASE AND POLYNUCLEOTIDE ENCODING SAME, AND METHOD FOR PRODUCING ISOPRENE MONOMER (57) The present invention provides means useful for (b) a polynucleotide that comprises a nucleotide se- establishing an excellent isoprene monomer production quence having 90% or more identity to the nucleotide system.
    [Show full text]
  • Inhibition of Hepatic Cholesterol Synthesis
    Proc. Nati Acad. Sci. USA Vol. 79, pp. 4873-4877, August 1982 Biochemistry Inhibition of hepatic cholesterol synthesis- in mice by sterols with shortened and stereochemically varied side chains [feedback regulation/3-hydroxy-3-methylglutaryl-coenzyme A reductase/mevalonic acid/(E)- and (Z)-17(20)-dehydrocholesterol/ pregn-5-en-3.&ol] KATHERINE A. ERICKSON* AND WILLIAM R. NESt Department ofBiological Sciences, Drexel University, Philadelphia, Pennsylvania 19104 Communicated by E. H. Ahrens, Jr., May 5, 1982 ABSTRACT Micewere fedcholesterol orvarious other sterols chain found in cholesterol. The literature provided no guidance for 26 hr, after which the amount ofhepatic cholesterol synthesis on the effect that this changewould have. The only steroids with was measured in a cell-free system. The following sterols were as shortened side chains previously examined (hormones and bile effective as cholesterol itself in, depressing the conversion of ace- acids) have had oxygen atoms on or in place of the side chain tate into sterol: pregn-5.en-3.&ol, which lacks an isohexyl group (10-12). Polar groups, ofcourse, would be expected to have a on C-20; (E)-17(2G)-dehydrocholesterol, in which the isohexyl marked effect in their own-right. In addition, many ofthe com- group is fixed to the right; (E)-20(22)-dehydrocholesterol, in which pounds-e.g., bile acids and testosterone-also had- changes C-23 is oriented away from the nucleus; and 20-epicholesterol. in the In order to make an exact comparison with cho- Moreover, when the isohexyl group was fixed to the left in (Z)- nucleus. 17(20)-dehydrocholesterol, this dietary sterol, identified in the lesterol we used the 3,(3hydroxy-A5-sterols (androst-5-en-3/-ol liver, causednotonly a depression in the conversion ofacetate into and pregn-5-en-3(-ol), which had no other polar groups and sterols but also-a depression in the conversion of both mevalonate zeroandtwocarbon atoms on C-17, respectively.
    [Show full text]
  • European Patent Office
    (19) & (11) EP 2 380 989 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 26.10.2011 Bulletin 2011/43 C12Q 1/26 (2006.01) C12N 1/20 (2006.01) C12N 9/02 (2006.01) (21) Application number: 10731334.8 (86) International application number: (22) Date of filing: 19.01.2010 PCT/JP2010/050565 (87) International publication number: WO 2010/082665 (22.07.2010 Gazette 2010/29) (84) Designated Contracting States: (72) Inventor: MATSUOKA, Takeshi AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Tokyo 101-8101 (JP) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR (74) Representative: Forstmeyer, Dietmar et al BOETERS & LIECK (30) Priority: 19.01.2009 JP 2009009177 Oberanger 32 80331 München (DE) (71) Applicant: Asahi Kasei Pharma Corporation Tokyo 101-8101 (JP) (54) METHOD AND REAGENT FOR DETERMINING MEVALONIC ACID, 3- HYDROXYMETHYLGLUTARYL-COENZYME A AND COENZYME A (57) The present invention provides a method for taryl coenzyme A in the presence of a hydrogen acceptor measuring the concentration of an analyte in a test so- X, a hydrogen donor Y, and coenzyme A; and (q) a step lution wherein the analyte is mevalonic acid and/or 3- of measuring an amount of: a reduced hydrogen acceptor hydroxymethylglutaryl coenzyme A, comprising the fol- X that is produced; or an oxidized hydrogen donor Y that lowing steps (p) and (q): (p) a step of allowing an enzyme is produced; or a hydrogen acceptor X that is decreased; that catalyzes a reaction represented by Reaction For- or a hydrogen donor Y that is decreased, wherein the mula 1 and an enzyme that catalyzes a reaction repre- hydrogen donor Y and the reduced hydrogen acceptor sented by Reaction Formula 2 to act on a test solution X are not the same.
    [Show full text]
  • Genetic Variants in Root Architecture-Related Genes in A
    Prince et al. BMC Genomics (2015) 16:132 DOI 10.1186/s12864-015-1334-6 RESEARCH ARTICLE Open Access Genetic variants in root architecture-related genes in a Glycine soja accession, a potential resource to improve cultivated soybean Silvas J Prince1†, Li Song1†, Dan Qiu1, Joao V Maldonado dos Santos1,2, Chenglin Chai1, Trupti Joshi2,3, Gunvant Patil1, Babu Valliyodan1, Tri D Vuong1, Mackensie Murphy1, Konstantinos Krampis4, Dominic M Tucker4, Ruslan Biyashev4, Anne E Dorrance5, MA Saghai Maroof4, Dong Xu2,3, J Grover Shannon1 and Henry T Nguyen1,2* Abstract Background: Root system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja. Results: Wild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (Dtypecyclinand auxin efflux carrier protein) with insertion/ deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean.
    [Show full text]