DOCSLIB.ORG

Cloning and Characterization of Sesquiterpene Lactone Synthases from Inula Britannica L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cloning and Characterization of Sesquiterpene Lactone Synthases from Inula Britannica L Cloning and characterization of sesquiterpene lactone synthases from Inula britannica L. Namraj Dhami January 20, 2012 Cloning and characterization of sesquiterpene lactone synthases from Inula britannica L. A thesis submitted for Master Degree in Plant Biotechnology, specialization in Plant Functional Genomics (PPH-80439) Namraj Dhami Student Regd. no. 760815181100 Supervisors Examiner Dr. Ir. Sander Vander Krol Prof. dr. Harro J. Bouwmeester Qing Liu Laboratory of Plant Physiology Wageningen University and Research Centre Droevendaalsesteeg 1, 6708 PB, Wageningen The Netherlands January, 2012 Abstract Inula britannica is a common traditional medicinal herb belonging to Asteraceae family. It is widely used in Traditional Chinese Medicine as anticancer, antibacterial, and antiinflammatory remedies. Neobritannilactone B, britannilactone and other sesquiterpene lactones (STLs) are biologically active compounds found in I. britannica. However, biosynthesis of STLs has not been studied in I. britannica yet. This work was carried out to clone and characterize sesquiterpene synthase genes and elucidate sesquiterpene lactone biosynthetic pathway in I. britannica. Disc floret cDNA library of I. britannica was analysed to find consensus expressed sequence tags (ESTs) of putative germacrene A synthase (GAS), germacrene A oxidase (GAO) and costunolide synthase (COS) candidate genes. Rapid amplification of cDNA ends (RACE) PCR was carried out to identify missing 3' and 5' end sequences of candidate genes. Open reading frames (ORFs) of putative candidate genes were determined by assembling sequences of respective RACE fragment and consensus ESTs. Coding sequences of putative IbGAS, IbGAO and IbCOS were isolated from I. britannica disc floret RNA and functional characterization was carried out by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana. Coding sequences were cloned in to pYEDP60 yeast expression vector and transformed in to yeast. Yeast cultures transformed with putative IbGAS, IbGAO and IbCOS candidates were analysed by GC-MS for targeted metabolites produced by putative genes. Similarly, for in planta characterization of gene function, cds of putative IbGAS, IbGAO and IbCOS candidates were cloned in to pBIN+ binary expression vector and subsequently agro-infiltrated in N. benthamiana along with Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase (AtHMGR) and farnesyl pyrophosphate synthase (AtFPS). Headspace and solvent extracts of agro-infiltrated N. benthamiana leaves were analysed by GC-MS. In planta expression assay of putative IbGAS revealed that germacrene A production was increased ~ 8 fold in N. benthamiana. Thus, putative IbGAS candidate gene was characterized as germacrene A synthase. However, function of IbGAS was not confirmed in yeast. On the other hand, putative IbGAO and IbCOS did not catalyse their respective substrates, both in yeast as well as in planta, to produce germacrene A acid and costunolide, respectively. This study is pioneer molecular work in I. britannica; hence it will certainly play a crucial role to unravel the biosynthetic pathway of pharmaceutically important neobritannilactone B, britannilactone and their derivatives. Keywords: Inula britannica, sesquiterpene lactone, neobritannilactone B, germacrene A synthase, germacrene A oxidase, costunolide synthase and heterologous expression. ii Acknowledgement It is my great pleasure to extend sincere gratitude to Dr. Ir. Sander van der Krol for his valuable suggestions and constant encouragement during supervision of research project. I appreciate generous guidance of Qing Liu during wet experiments and data analysis. I am also thankful to Miriam Goedbloed for manifold help and suggestions during wet experiments, Danijela Misic for helping during agro-infiltration and extraction, and Francel Verstappen for help in chromatographic analysis. I would also like to express sincere thanks to Prof. Harro J. Bouwmeester for making arrangements for my research work in the laboratory of plant physiology. I am very much thankful to Netherlands organization for international cooperation in higher education (nuffic) for granting fellowship to study MSc Plant Biotechnology in Wageningen UR and grateful to Pokhara University, Nepal for providing leaves to study abroad. I would like to use this opportunity to express my sincere gratitude and due respect to reverend parents, Keshab Singh Dhami and Durga Devi Dhami; as well as elder brother Er. Govind Singh Dhami; for everlasting encouragement and inspiration that hold me on academic career. I would like to admire constant care and support of my beloved wife Janki and love of sweet daughter; Opal. Last but not the least; I would like to mention care and compliments of bhauju; Ishwari, younger brother; Umesh, and cousins; Milan and Sijan that really worked upon me to persevere here in Wageningen. Namraaj Dhami iii Contents Abstract ........................................................................................................................................ ii Acknowledgement ...................................................................................................................... iii List of tables ............................................................................................................................... vi List of figures ............................................................................................................................. vii 1. INTRODUCTION ........................................................................................................................ 1 1.1 Inula britannica: a traditional medicinal herb ....................................................................... 1 1.2 Phytochemistry of I. britannica ............................................................................................. 1 1.3 Biosynthesis of sesquiterpene lactones in I. britannica ......................................................... 2 2. METHODOLOGY ....................................................................................................................... 4 2.1 Identification of sesquiterpene synthase candidates in Inula britannica ............................... 4 2.1.1 Analysis of Inula britannica disc floret cDNA library ................................................... 4 2.1.2 Primer design for RACE PCR ........................................................................................ 4 2.1.3 First strand RACE ready cDNA synthesis ..................................................................... 4 2.1.4 RACE PCR for Inula britannica candidate genes .......................................................... 4 2.1.5 Cloning of RACE fragments and determination of open reading frame ........................ 5 2.1.6 Isolation of full length candidate genes .......................................................................... 5 2.2 Functional characterization of Inula britannica candidate genes in yeast ............................. 6 2.2.1 Cloning of candidate cds in yeast expression vector ...................................................... 6 2.2.2 Expression of Inula britannica candidate genes in yeast ............................................... 6 2.2.3 GC-MS analysis of transgenic yeast extracts ................................................................. 7 2.3 Functional characterization of Inula britannica candidate genes in Nicotiana benthamiana 7 2.3.1 Cloning of Inula britannica candidate gene in Gateway® entry vector ......................... 7 2.3.2 Cloning of Inula britannica candidate genes in binary expression vector ..................... 8 2.3.3 Transformation of Agrobacterium tumefaciens .............................................................. 8 2.3.4 Agro-infiltration of Nicotiana benthamiana with Inula britannica candidate genes ..... 8 2.3.5 Headspace analysis of agro-infiltrated Nicotiana benthamiana leaves .......................... 9 2.3.6 GC- MS analysis of agro-infiltrated Nicotiana benthamiana leaves .............................. 9 3. RESULTS AND DISCUSSION ................................................................................................ 11 3.1 Disc florets of Inula britannica are right targets for sesquiterpene synthase gene isolation 11 3.2 Isolation and characterization of Inula britannica germacrene A synthase (IbGAS) .......... 11 3.2.1 Putative IbGAS identified from Inula britannica ......................................................... 11 3.2.2 Putative IbGAS increased germacrene A production in planta .................................... 12 3.2.3 Putative IbGAS not active in yeast ............................................................................... 13 3.3 Isolation and characterization of Inula britannica germacrene A oxidase (IbGAO) ........... 14 iv 3.3.1 Putative IbGAO identified from Inula britannica ........................................................ 14 3.3.2 Putative IbGAO cannot utilize germacrene A as a substrate ........................................ 15 3.4 Identification and characterization of Inula britannica costunolide synthase (IbCOS) ....... 16 3.4.1 Putative IbCOS identified from Inula britannica ......................................................... 16 3.4.2 Function of putative IbCOS not determined in yeast and in planta .............................. 16 3.5 Conclusion ..........................................................................................................................
Load more

Recommended publications
  • The Biosynthesis of Sesquiterpene Lactones in Chicory (Cichorium Intybus L.) Roots Promotor Prof
    The Biosynthesis of Sesquiterpene Lactones in Chicory (Cichorium intybus L.) Roots Promotor Prof. dr. Æ. de Groot, hoogleraar in de bio-organische chemie, Wageningen Universiteit Co-promotoren Dr. M.C.R. Franssen, universitair hoofddocent, Laboratorium voor Organische Chemie, Wageningen Universiteit Dr. ir. H.J. Bouwmeester, senior onderzoeker, Business Unit Celcybernetica, Plant Research International Promotiecommissie Prof. dr. J. Gershenzon (Max Planck Institute for Chemical Ecology, Germany) Prof. dr. L.H.W. van der Plas (Wageningen Universiteit) Prof. dr. ir. I.M.C.M. Rietjens (Wageningen Universiteit) Prof. dr. E.J.R. Sudhölter (Wageningen Universiteit) Jan-Willem de Kraker The Biosynthesis of Sesquiterpene Lactones in Chicory (Cichorium intybus L.) Roots Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit prof. dr. ir. L. Speelman in het openbaar te verdedigen op woensdag 9 januari 2002 des namiddags te vier uur in de Aula de Kraker, J.-W. The Biosynthesis of Sesquiterpene Lactones in Chicory (Cichorium intybus L.) Roots Thesis Wageningen University – with summaries in English and Dutch ISBN 90-5808-531-7 Voorwoord — Preface De afgelopen vijf jaar was het voor mij witlof, dag in dag uit. Waarschijnlijk is dit ook de reden dat ik al ruim 3 jaar geen witlof meer kan ruiken, laat staan eet. Als je dan ongeacht waar je in Europa bent overal in de wegberm uitsluitend blauwe bloemen ziet opduiken, is het wel goed en verstandig er eens een punt achter te zetten. Hoewel het eind van dit promotie- onderzoek door al het ‘wachtgeldgedoe’ en bijbehorende ‘dode mussen’ wel erg abrupt en weinig chique kwam, kan ik me nu met goed gevoel weer eens op wat anders storten.
    [Show full text]
  • Production De Terpènes Fonctionnalisés Par Les Cytochromes P450 De Plantes Recombinants
    Université de Strasbourg Ecole doctorale des Sciences de la Vie et de la Santé (ED 414) Institut de Biologie Moléculaire des Plantes (CNRS, UPR2357) THESE Présentée pour obtenir le grade de DOCTEUR de l’Université de Strasbourg Discipline : Biochimie et biologie moléculaire par Carole Gavira Production de terpènes fonctionnalisés par les cytochromes P450 de plantes recombinants Soutenue le 26 février 2013 à Strasbourg devant le jury composé de : Dr. D. Werck-Reichhart Directeur de Recherche au CNRS, IBMP, Strasbourg Directeur de thèse Pr. F. Bourgaud Professeur à l’ENSAIA de Nancy Rapporteur Pr. A. Tissier Professeur à l’IPB Leibniz Institut, Halle, Allemagne Rapporteur Pr. T. Bach Professeur à l’Université de Strasbourg Examinateur Dr. R. Feyereisen Directeur de Recherche à l’INRA, Sophia-Antipolis Examinateur Remerciements J’adresse mes plus sincères remerciements à Mme Danièle Werck-Reichhart, ma Directrice de thèse, pour m’avoir donné l’opportunité de réaliser ces travaux et pour la confiance et la liberté qu’elle m’a accordée. Je remercie également M. Joseph Zucca, mon co- Directeur, et Mme Fanny Lambert, sa Collaboratrice, de la Société V. MANE Fils, qui ont été les initiateurs de ce projet réalisé en harmonie sous convention CIFRE, et qui m’ont permis de participer à deux congrès de renommée internationale. Je souhaite, d’une part, remercier vivement M. Thomas Bach, M. Frédéric Bourgaud, M. Alain Tissier et M. René Feyereisen d’avoir accepté d’être membres de mon jury. Je voudrais, d’autre part, remercier M. Christophe Crocoll, pour son aide précieuse et sa disponibilité, M. Erich Glawischnig et Mme Laurence Miesch pour leur collaboration.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 7,214.507 B2 Bouwmeester Et Al
    US007214507B2 (12) United States Patent (10) Patent No.: US 7,214.507 B2 BOuWmeester et al. (45) Date of Patent: May 8, 2007 (54) PLANT ENZYMES FOR BIOCONVERSION (56) References Cited (75) Inventors: Hendrik Jan Bouwmeester, Renkum U.S. PATENT DOCUMENTS (NL); Jan-Willem de Kraker, Jena 6,200,786 B1* 3/2001 Huang et al. ............... 435,132 (DE); Marloes Schurink, Ede (NL); 6,451,576 B1* 9/2002 Croteau et al. ............. 435/232 Raoul John Bino, Wageningen (NL); Aede de Groot, Wageningen (NL); OTHER PUBLICATIONS Maurice Charles R. Franssen, Dedeyan B et al. 2000. Appl Env Microbiol 66: 925-929.* Wageningen (NL) Yoder OC et al. 2001. Curr Opin Plant Biol 4: 315-321.* Nielsen KAetal. 2005. Cytochrome P450s in plants. In Cytochrome (73) Assignee: Plant Research International B.V., P450: structure, mechanism, and biochemistry, 3' ed., Ortiz de Wageningen (NL) Montellano, ed., p. 553.* Bohlmann, Jorg, et al., “Plant terpenoid synthases: Molecular (*) Notice: Subject to any disclaimer, the term of this biology and phylogenetic analysis”. Proc. Natl. Acad. Sci. 1998, patent is extended or adjusted under 35 95:4126-4133. de Kraker, Jan-Willem, et al., “Biosynthesis of Germacrene A U.S.C. 154(b) by 0 days. Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP-Dependent (21) Appl. No.: 10/489,762 Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis”. Plant Physiology 2001, 125:1930-1940. (22) PCT Filed: Sep. 17, 2002 de Kraker, Jan-Willem, et al., “Germacrenes from fresh costus roots”, Phytochemistry 2001, 58:481-487. (86). PCT No.: PCT/NLO2/OO591 de Kraker, Jan-Willem, et al., “(+)-Germacrene A Biosynthesis'.
    [Show full text]
  • Investigating Labdane-Related Diterpenoids Biosynthetic Network In
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Investigating labdane-related diterpenoids biosynthetic network in rice: characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase Yisheng Wu Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Wu, Yisheng, "Investigating labdane-related diterpenoids biosynthetic network in rice: characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase" (2012). Graduate Theses and Dissertations. 12680. https://lib.dr.iastate.edu/etd/12680 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigating labdane-related diterpenoids biosynthetic network in rice: Characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase by Yisheng Wu A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Reuben Peters, Major Professor Gustavo MacIntosh Thomas Bobik Bing Yang Mark Hargrove Iowa State University Ames, Iowa 2012 Copyright © Yisheng Wu , 2012. All
    [Show full text]
  • Elucidation of the Sesquiterpene Lactone Biosynthetic Pathway in Feverfew (Tanacetum Parthenium)
    Elucidation of the sesquiterpene lactone biosynthetic pathway in feverfew (Tanacetum parthenium) Qing Liu Thesis committee Promotor Prof. dr. ir. H.J. Bouwmeester Professor of Plant Physiology Wageningen University Co-promotor Dr. S.V.D. Krol Associate professor, Laboratory of Plant Physiology Wageningen University Other members Prof. dr. A. Ferrer, Centre for Research in Agricultural Genomics, Bellaterra, Spain Dr. M.C.R. Franssen, Wageningen University Prof. dr. M.E. Schranz, Wageningen University Dhr. dr. ir. R.C. Schuurink, University of Amsterdam This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Elucidation of the sesquiterpene lactone biosynthetic pathway in feverfew (Tanacetum parthenium) Qing Liu Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 2nd December 2013 at 1:30 p.m. in the Aula. Qing Liu Elucidation of the sesquiterpene lactone biosynthetic pathway in feverfew (Tanacetum parthenium) 134 pages PhD thesis, Wageningen University, Wageningen, the Netherlands (2013) With summaries in English and Dutch ISBN 978-94-6173-756-4 Contents Chapter 1 General introduction 7 Chapter 2 Biosynthesis and localization of parthenolide in glan- dular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.) 19 Chapter 3 Reconstitution of the costunolide biosynthetic
    [Show full text]
  • Cytochrome P450 from Plants: Platforms for Valuable Phytopharmaceuticals
    Villa-Ruano et al Tropical Journal of Pharmaceutical Research April 2015; 14 (4): 731-742 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v14i4.24 Review Article Cytochrome P450 from Plants: Platforms for Valuable Phytopharmaceuticals Nemesio Villa-Ruano1*, Yesenia Pacheco-Hernández2, Edmundo Lozoya- Gloria3, Carlos Jonathan Castro-Juárez1, Clemente Mosso-Gonzalez1 and Sergio Alberto Ramirez-Garcia1 1Universidad de la Sierra Sur. Guillermo Rojas Mijangos S/N, C.P. 70805, Ciudad Universitaria, Miahuatlán de Porfirio Díaz, 2Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional–Unidad Oaxaca. Hornos 1003, Col. Noche Buena, C.P. 71230 Sta. Cruz Xoxocotlán, Oaxaca, 3Centro de Investigación y de Estudios Avanzados del I.P.N. Km 9.6, Libramiento Norte Carretera Irapuato-León, C.P. 36821, Irapuato, Gto., México *For correspondence: Email: [email protected] Received: 14 December 2014 Revised accepted: 16 March 2015 Abstract Cytochrome P450 enzymes are important for biotechnology due to their capacity to modify diverse secondary metabolites that may produce chemicals with pharmacological properties. Most terpenes, flavonoids and alkaloids require P450 catalytic functions to reach their biological activity. In the last ten years, several efforts have focused on the expression and production of these three main types of secondary metabolites in engineered microorganisms and plants using P450 of ethnobotanical origin. Despite this, several P450 coding sequences from plant sources are discovered yearly but only a few have been screened by functional genomics. Amongst them, only a few have shown potentials for use in sustainable production of novel drugs and highly valuable products.
    [Show full text]
  • Asteraceae) Family
    University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2013-05-06 Biochemical and evolutionary studies of sesquiterpene lactone metabolism in the sunflower (Asteraceae) family Nguyen, Trinh Don Nguyen, T. D. (2013). Biochemical and evolutionary studies of sesquiterpene lactone metabolism in the sunflower (Asteraceae) family (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25121 http://hdl.handle.net/11023/702 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Biochemical and evolutionary studies of sesquiterpene lactone metabolism in the sunflower (Asteraceae) family by Trinh Don Nguyen A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA APRIL, 2013 © Trinh Don Nguyen 2013 Abstract Plants have evolved the capacity to synthesize a myriad of specialized metabolites which enhance their fitness in specific living conditions. These compounds are also widely utilized for human purposes. Elucidating the enzymes in plant specialized metabolism has been one of the main forces driving plant biochemistry. The more intriguing question, however, is how these enzymes evolved to acquire their existent functions. The Asteraceae, the largest flowering plant family, is well-known for its enormously diverse and lineage-characteristic contents of sesquiterpene lactones (STLs).
    [Show full text]
  • Kauniolide Synthase Is a P450 with Unusual Hydroxylation and Cyclization-Elimination Activity
    ARTICLE DOI: 10.1038/s41467-018-06565-8 OPEN Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity Qing Liu1,2, Arman Beyraghdar Kashkooli1, David Manzano3,4, Irini Pateraki 2, Lea Richard1, Pim Kolkman1, Maria Fátima Lucas5, Victor Guallar5,6, Ric C.H. de Vos7, Maurice C.R. Franssen8, Alexander van der Krol 1 & Harro Bouwmeester 1,9 Guaianolides are an important class of sesquiterpene lactones with unique biological and 1234567890():,; pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kaunio- lide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines ste- reoselective hydroxylation of costunolide at the C3 position, with water elimination, cycli- zation and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones. 1 Laboratory of Plant Physiology, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. 2 Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871, Denmark. 3 Plant Metabolism and Metabolic Engineering Program, Centre for Research in Agricultural Genomics (CRAG) (CSIC-IRTA-UAB-UB), 08193, Barcelona, Spain. 4 Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, Campus Diagonal, Av.
    [Show full text]
  • Kauniolide Synthase Is a P450 with Unusual Hydroxylation and Cyclization-Elimination Activity
    UvA-DARE (Digital Academic Repository) Kauniolide synthase is a P450 with unusual hydroxylation and cyclization- elimination activity Liu, Q.; Beyraghdar Kashkooli, A.; Manzano, D.; Pateraki, I.; Richard, L.; Kolkman, P.; Lucas, M.F.; Guallar, V.; de Vos, R.C.H.; Franssen, M.C.R.; van der Krol, A.; Bouwmeester, H. DOI 10.1038/s41467-018-06565-8 Publication date 2018 Document Version Final published version Published in Nature Communications License CC BY Link to publication Citation for published version (APA): Liu, Q., Beyraghdar Kashkooli, A., Manzano, D., Pateraki, I., Richard, L., Kolkman, P., Lucas, M. F., Guallar, V., de Vos, R. C. H., Franssen, M. C. R., van der Krol, A., & Bouwmeester, H. (2018). Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity. Nature Communications, 9, [4657]. https://doi.org/10.1038/s41467-018-06565-8 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands.
    [Show full text]
  • Dependent Monooxygenases
    The evolution of the flavin-dependent monooxygenases Chopping and Changing: the Evolution of the Flavin- dependent Monooxygenases Maria Laura Mascotti1*, Maximiliano Juri Ayub1, Nicholas Furnham2, Janet M. Thornton3, Roman A Laskowski3* 1IMIBIO-SL CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, San Luis, D5700HHW, Argentina 2Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK 3EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK *corresponding authors: Maria Laura Mascotti ([email protected]), Roman A. Laskowski ([email protected]) Supplementary Data Page nº Table S1 …………………….. 2-7 Dataset S1 ………………………. 8 Table S2 ………………………. 9 Figure S1 ……………………... 10 Figure S2 ……………………... 11 References ..……………………. 12 1 The evolution of the flavin-dependent monooxygenases Table S1 Classification of enzymes included in the EC sub-subclasses 1.13.12, 1.14.13 and 1.14.14. Data collected from the enzyme database BRENDA (http://www.brenda-enzymes.org/, last accessed on 8th June 2016). The classes containing flavin- dependent monooxygenases are identified by FMO in the final column and their class (A-H). EC PDBs Enzyme Name Classification* 1.13.12.1 - arginine 2-monooxygenase FMO G 1.13.12.2 - lysine 2-monooxygenase FMO G 1.13.12.3 2 tryptophan 2-monooxygenase FMO G 1.13.12.4 30 lactate 2-monooxygenase FMO H 1.13.12.5 11 Renilla-luciferin 2-monooxygenase Luc 1.13.12.6 - Cypridina-luciferin 2-monooxygenase
    [Show full text]
  • (19) United States (12) Patent Application Publication (10) Pub
    US 20130244920A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2013/0244920 A1 Lee et al. (43) Pub. Date: Sep. 19, 2013 (54) WATER SOLUBLE COMPOSITIONS (52) US. Cl. INCORPORATING ENZYMES, AND METHOD USPC ......................................... .. 510/392; 264/299 OF MAKING SAME (57) ABSTRACT (76) Inventors: David M. Lee, CroWn Point, IN (US); Jennifer L‘ Sims’ Lowell’ IN (Us) Disclosed herein are Water soluble compositions, such as ?lms, including a mixture of a ?rst Water-soluble resin, an (21) Appl' NO': 13/422’709 enzyme, and an enzyme stabilizer Which comprises a func (22) Filed: Man 16, 2012 tional substrate for the enzyme, methods of making such compositions, and methods of using such compositions, e.g. Publication Classi?cation to make packets containing functional ingredients. The enzymes can include proteases and mixtures of proteases (51) Int. Cl. With other enzymes, and the compositions provide good C11D 3/386 (2006.01) retention of enzyme function following ?lm processing and B29C 39/02 (2006.01) storage. US 2013/0244920 A1 Sep. 19,2013 WATER SOLUBLE COMPOSITIONS preheated to a temperature less than 77° C., optionally in a INCORPORATING ENZYMES, AND METHOD range ofabout 66° C. to about 77° C., or about 74° C.; drying OF MAKING SAME the Water from the cast mixture over a period of less than 24 hours, optionally less than 12 hours, optionally less than 8 FIELD OF THE DISCLOSURE hours, optionally less than 2 hours, optionally less than 1 [0001] The present disclosure relates generally to Water hour, optionally less than 45 minutes, optionally less than 30 soluble ?lms.
    [Show full text]
  • Thesis Revision 12.8.17
    Engineering microbial production of terpenoids by Jeff Wong A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant and Microbial Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Jay D. Keasling, Co-chair Professor Henrik V. Scheller, Co-chair Professor Krishna Niyogi Professor John Dueber Fall 2017 Engineering microbial production of terpenoids Copyright 2017 by Jeff Wong Abstract Engineering microbial production of terpenoids by Jeff Wong Doctor of Philosophy in Plant and Microbial Biology University of California, Berkeley Professors Jay D. Keasling and Henrik V. Scheller, Co-chairs Terpenoids are a broad class of natural products that have applications in medicine as well as commodity chemicals and biofuels. However, many terpenoids are sourced from field grown plants, which suffer from inconsistent yield and composition. Additionally, many terpenoid biosynthetic pathways are poorly understood or completely uncharacterized due to limitations regarding genetic resources and heterologous expression. Recent improvements in synthetic biology such as DNA synthesis, targeted genome editing, and next generation sequencing have enabled accelerated research and development in this field. In this work I will describe my contributions to the characterization of two plant-derived medicinal terpenoid pathways: the prostratin and valerenic acid biosynthetic pathways. Chapter 1 begins with a discussion of the challenges and successes associated with engineering terpenoid production in yeast as well as other commonly engineered microbes. This chapter covers their applications in medicine, biofuels and other commodity chemicals. Chapter 2 presents my work in developing transcriptomic resources for identifying P450s and other candidate genes involved in the biosynthesis of prostratin.
    [Show full text]