Final Thesis Elucidation of the product synthesis of the sesquiterpene synthase Cop6 isolated from Coprinus cinereus Marie Andersson Final Thesis carried out at University of Minnesota 2009-03-12 LITH-IFM-x-EX--09/2039--SE Department of Physics, Chemistry and Biology Linkoping University SE-581 83 Linkoping, Sweden Department of Physics, Chemistry and Biology Division of Molecular Biotechnology Elucidation of the product synthesis of the sesquiterpene synthase Cop6 isolated from Coprinus cinereus Marie Andersson Final Thesis carried out at University of Minnesota 2009-03-12 Supervisors Claudia Schmidt-Dannert Fernando Lopez Gallego Examiner Maria Sunnerhagen Upphovsrätt Detta dokument hålls tillgängligt på Internet – eller dess framtida ersättare – under 25 år från publiceringsdatum under förutsättning att inga extraordinära omständigheter uppstår. Tillgång till dokumentet innebär tillstånd för var och en att läsa, ladda ner, skriva ut enstaka kopior för enskilt bruk och att använda det oförändrat för ickekommersiell forskning och för undervisning. Överföring av upphovsrätten vid en senare tidpunkt kan inte upphäva detta tillstånd. All annan användning av dokumentet kräver upphovsmannens medgivande. För att garantera äktheten, säkerheten och tillgängligheten finns lösningar av teknisk och administrativ art. Upphovsmannens ideella rätt innefattar rätt att bli nämnd som upphovsman i den omfattning som god sed kräver vid användning av dokumentet på ovan beskrivna sätt samt skydd mot att dokumentet ändras eller presenteras i sådan form eller i sådant sammanhang som är kränkande för upphovsmannens litterära eller konstnärliga anseende eller egenart. För ytterligare information om Linköping University Electronic Press se förlagets hemsida http://www.ep.liu.se/. Copyright The publishers will keep this document online on the Internet – or its possible replacement – for a period of 25 years starting from the date of publication barring exceptional circumstances. The online availability of the document implies permanent permission for anyone to read, to download, or to print out single copies for his/hers own use and to use it unchanged for non-commercial research and educational purpose. Subsequent transfers of copyright cannot revoke this permission. All other uses of the document are conditional upon the consent of the copyright owner. The publisher has taken technical and administrative measures to assure authenticity, security and accessibility. According to intellectual property law the author has the right to be mentioned when his/her work is accessed as described above and to be protected against infringement. For additional information about the Linköping University Electronic Press and its procedures for publication and for assurance of document integrity, please refer to its www home page: http://www.ep.liu.se/. © Marie Andersson Abstract Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom Coprinus cinereus that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly α- cuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type. Table of contents 1 Introduction ........................................................................................................................ 7 1.1 Background ................................................................................................................ 7 1.1.1 Mushrooms as nature products........................................................................... 7 1.1.2 Terpenoids.......................................................................................................... 7 1.1.3 Sesquiterpene cyclases ....................................................................................... 8 1.2 Aim............................................................................................................................. 8 2 Theory ................................................................................................................................9 2.1 Trichodiene synthase.................................................................................................. 9 2.1.1 Modelling ........................................................................................................... 9 2.1.2 Structure-Based Mechanism ............................................................................ 11 2.1.3 NSE/DTE motif................................................................................................ 12 2.1.4 Arginine-rich domain (DRRYR)...................................................................... 12 2.1.5 Aspartate-rich domain (DDXX(D,E)).............................................................. 13 2.2 Methods.................................................................................................................... 14 2.2.1 Site directed mutagenesis using PCR............................................................... 14 2.2.2 Cloning and transformation.............................................................................. 15 2.2.3 Purification....................................................................................................... 16 2.2.4 GC-MS ............................................................................................................. 16 3 Experimental details......................................................................................................... 18 3.1 Modelling ................................................................................................................. 18 3.2 Site directed mutagenesis......................................................................................... 18 3.2.1 Y195V and D102E........................................................................................... 18 3.2.2 I74M and N224D ............................................................................................. 18 3.2.3 V98X Library ................................................................................................... 19 3.3 Agarose gel electrophoresis ..................................................................................... 20 3.4 Cloning..................................................................................................................... 20 3.5 Transformation......................................................................................................... 21 3.6 General growth of E. coli ......................................................................................... 21 3.7 DNA extraction ........................................................................................................ 21 3.8 Expression of protein ............................................................................................... 21 3.9 GC-MS ..................................................................................................................... 22 3.10 Immobilized Metal Affinity Chromatography (IMAC)........................................... 22 3.11 Hydrophobic interaction chromatography (HIC)..................................................... 22 3.12 Desalting and concentration..................................................................................... 23 3.13 SDS-PAGE............................................................................................................... 23 3.14 Protein concentration determination ........................................................................ 23 3.15 Kinetic parameters.................................................................................................... 23 4 Results .............................................................................................................................. 24 4.1 Work process............................................................................................................ 24 4.2 Identification of target sites...................................................................................... 24 4.3 Products by Cop6 mutants........................................................................................ 26 4.3.1 Products by Cop6 single mutants..................................................................... 26 4.3.2 Products by Cop6 V98X library mutants ......................................................... 27 4.4 Kinetic parameters.................................................................................................... 29 4.5 Purification of Cop6