DOCSLIB.ORG

Kauniolide Synthase Is a P450 with Unusual Hydroxylation and Cyclization-Elimination Activity

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:26 Sep 2021 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. de Joan XXIII, 27-31, 08028 Barcelona, Spain. 5 Barcelona Supercomputing Center (BSC), C/ Jordi Girona 29, 08034 Barcelona, Spain. 6 ICREA, Pg Lluís Companys 23, 08010 Barcelona, Spain. 7 Wageningen Plant Research, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. 8 Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands. 9 Plant Hormone Biology group, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands. These authors contributed equally: Qing Liu, Arman Beyraghdar Kashkooli. Correspondence and requests for materials should be addressed to A.B.K. (email: [email protected]) or to H.B. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4657 | DOI: 10.1038/s41467-018-06565-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06565-8 esquiterpene lactones are C15 terpenoids and constitute a that kauniolide biosynthesis does not require a cyclase as sug- Smajor class of plant secondary metabolites with diverse gested before. The full kauniolide biosynthesis pathway is suc- chemical structures. They are present in plant species in the cessfully reconstructed in both the heterologous hosts Nicotiana Acanthaceae, Apiaceae and Asteraceae1 with over 4000 different benthamiana and yeast. Modelling is used to support elucidation structures reported so far2. Sesquiterpene lactones are classified in of the putative enzymatic steps catalysed by KLS. The isolation of six bicyclic or tricyclic classes named guianolides, pseudoguaia- the KLS gene opens up opportunities for metabolic engineering of nolides, xanthanolides, eremophilanolides, eudesmanolides and guaianolide sesquiterpene lactones. germacranolides3. Naturally occurring sesquiterpene lactones α γ α β contain an -methylene- -butyrolactone or , -unsaturated Results cyclopentenone moiety. These, or more in general the the α, β- CYP71 activity on costunolide and parthenolide. In feverfew unsaturated carbonyl, largely account for their biological activity extracts, multiple costunolide- and parthenolide-derived products against cancer and inflammation3,4. Guaianolides are defined by are detected9. Previously, it was shown that feverfew P450s acting their special 5-7-5 tricyclic structure5 and these compounds are of on germacrene A, germacrene A acid, costunolide and parthe- interest because of their biological activity against prostate can- – nolide all belong to the CYP71 family11 14,18. Moreover, the cer6, anti-mycobacteria activity7, inhibition of parasite Trypano- genes encoding the enzymes of the parthenolide biosynthesis soma cruzi growth8 and human antioxidant response element pathway (TpGAS13, TpGAO13, TpCOS13 and TpPTS19) show a activation at low concentrations9. The biosynthetic pathways of a distinct expression profile during feverfew ovary development, number of important germacranolide sesquiterpene lactones have matching with the accumulation profile of parthenolide11,13,14 been elucidated. For instance, the biosynthesis of costunolide and (Supplementary Fig. 1a). The costunolide 3β-hydroxylase19, parthenolide has been elucidated by isolation and characteriza- though, displays a slightly different expression profile (Supple- tion of the germacrene A synthase (GAS)10,11, germacrene A mentary Fig. 1a and 1b). For our screen we therefore targeted oxidase (GAO)12, costunolide synthase (COS)13 and parthenolide CYP71 P450s with an expression profile similar to those of the synthase (PTS)14. In contrast, at present, enzymes involved in parthenolide biosynthesis pathway genes. P450 sequences were guaianolide formations have not been identified, preventing obtained by 454 sequencing of a trichome-enriched com- metabolic engineering of these classes of sesquiterpene lactones. plementary DNA (cDNA) library (www.terpmed.eu). Expression There are some hints about the biosynthesis of guaianolides: in profiling was done by Illumina RNA-sequencing of six different the family of Asteraceae and Apiaceae (e.g., feverfew (Tanacetum stages of feverfew ovary development. In total, 59 P450s of the parthenium), chicory (Cichorium intybus), thapsia (Thapsia gar- CYP71 family were detected of which 27 showed differential ganica)), two classes of sesquiterpene lactones, germacranolides expression during ovary development (Supplementary Fig. 1a). and guaianolides, often occur together4, suggesting that the bio- Cluster analysis of these 27 candidate P450s with the parthenolide synthesis of guaianolides may be related to that of germacrano- biosynthesis pathway genes resulted in a further selection of five lides. Moreover, root extracts of chicory can convert costunolide uncharacterized P450s for which expression in ovaries shows to the guaianolide leucodin2. This bioconversion could be clustering with those of the parthenolide biosynthesis pathway inhibited by a range of chemical P450 enzyme activity inhibitors2, genes (Supplementary Fig. 1a). For four of these uncharacterized suggesting that one or more P450 enzymes are involved in the P450s (Tp9025, Tp4149, Tp8879 and Tp8886), their expression conversion of costunolide to leucodin2. On the other hand, che- profile was independently validated by quantitative real-time mical synthesis showed that one guaianolide, kauniolide, could be reverse transcription–polymerase chain reaction (RT-qPCR), formed from parthenolide (Fig. 1b)15. In plants, parthenolide is confirming the RNA-sequencing data analysis in that they display produced from costunolide through the action of a P450 mono- an expression profile that is similar to the bona fide pathway oxygenase14. This implies that biosynthesis
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]
  • 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]
  • Generate Metabolic Map Poster
    An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. NisylCyc: Nicotiana sylvestris Cellular Overview Connections between pathways are omitted for legibility. Author: Aureliano Bombarely, Virginia Tech Clathrin Sec-independent heavy chain 2/ protein /Phytozome: Chloride translocase Bradi4g25570.1; AP-2 complex subunit conductance AP-2 complex protein TATA, Symbols: NDH- non-specific serine/ sigma//Phytozome: Clathrin regulatory protein subunit sigma/ Symbols: | chloroplastic/ O | NAD(P) threonine protein GRMZM2G139624_P01; light chain 1/ ICln//Symbols: /Symbols: | Major facilitator Plasma /Symbols: | H:plastoquinone kinase; Species: phenylalanine ammonia- /Symbols: | | nucleotide- SNARE-like superfamily membrane- Sulfate Sodium/ Sodium/ Bacterial sec- dehydrogenase Sodium/ Brachypodium Sulfate Sulfate Proton lyase; Species: Zea Clathrin light sensitive chloride superfamily protein | associated ORF transporter ORF ORF hydrogen hydrogen independent complex subunit O hydrogen distachyon; Gene: transporter transporter ORF pump- mays mays; Gene: chain protein | conductance protein | chr2:7383194- cation- 1.3 exchanger 2 exchanger 2 translocation
    [Show full text]