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Enlarging the Product Spectrum of Fatty Acid Synthases Enlarging the Product Spectrum of Fatty Acid Synthases Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie (FB 14) der Johann Wolfgang Goethe -Universität in Frankfurt am Main von Jan Gajewski aus Kirchheimbolanden Frankfurt 2017 (D30) vom Fachbereich Biochemie, Chemie und Pharmazie (FB 14) der Johann Wolfgang Goethe -Universität als Dissertation angenommen Dekan: Prof. Dr. Michael Karras 1. Gutachter: Prof. Dr. Martin Grininger 2. Gutachter: Prof. Dr. Eckhard Boles Datum der Disputation: 15.05.2017 Table of content 1 Deutsche Zusammenfassung ............................................................................... I 1.1 Hintergrund ................................................................................................... I 1.2 Rationales Engineering von FAS ................................................................. III 1.3 Fazit ........................................................................................................... VII 2 Introduction .......................................................................................................... 1 2.1 Fatty acid synthases ..................................................................................... 1 2.2 Structural aspects of type I FAS ................................................................... 2 2.2.1 Similarity to polyketide synthase (PKS) systems ................................... 4 2.3 Technological and industrial usage of FA ..................................................... 7 2.4 Rational engineering of FAS to broaden the product spectrum .................. 10 2.4.1 KS ....................................................................................................... 11 2.4.2 MPT .................................................................................................... 13 2.4.3 AT ....................................................................................................... 14 2.4.4 ACP ..................................................................................................... 15 2.4.5 TE ....................................................................................................... 16 2.4.1 Engineering FAS for the production of a polyketide ............................ 17 2.4.2 FAS as part of an in vivo metabolic pathway....................................... 17 3 Materials and methods ...................................................................................... 20 3.1 In vitro studies on C. ammoniagenes FAS ................................................. 20 3.1.1 Initial cloning ....................................................................................... 20 3.1.2 Point mutations ................................................................................... 21 3.1.3 Expression .......................................................................................... 23 3.1.4 Protein purification .............................................................................. 24 3.1.5 Activity assay of regular FAS .............................................................. 24 3.1.6 Activity assay of module 2 FAS (lactone producing FAS) ................... 25 3.1.7 Product assay for FA product distribution............................................ 25 3.1.8 Product assay for module 2 FAS (lactone production) ........................ 26 3.1.9 HPLC quantification ............................................................................ 26 3.1.1 Molecular dynamics simulations and kinetic model of FAS ................. 28 3.2 In vivo studies of short FA production in S. cerevisiae ............................... 28 3.2.1 Description yeast strain ....................................................................... 28 3.2.2 Vector description ............................................................................... 28 3.2.3 Primers ................................................................................................ 29 3.2.4 Transformation .................................................................................... 29 3.2.5 Cultures for product analysis ............................................................... 30 3.2.6 Sample processing .............................................................................. 30 3.2.7 Determination of free FA by gas chromatography (GC) ...................... 31 3.2.8 Metabolite analysis by HPLC .............................................................. 31 4 Results .............................................................................................................. 33 4.1 Engineering binding channels for chain length control ............................... 33 4.1.1 Engineering the KS domain................................................................. 34 4.1.1 Engineering the transferase domains .................................................. 42 4.1.1 Additional engineering of the KS domain ............................................ 47 4.2 Modulation of domain-domain interactions ................................................. 49 4.3 Engineering FAS for the production of a polyketide ................................... 55 4.4 Production of short FA in vivo using S. cerevisiae ...................................... 59 4.4.1 Testing KS domain mutations in vivo .................................................. 60 4.4.2 Testing of the transferase mutations in vivo ........................................ 61 4.4.3 Combinations of mutations in vivo ....................................................... 62 4.4.4 Examining cell viability ........................................................................ 63 5 Discussion ......................................................................................................... 67 5.1 Settings for probing biotechnological engineering efforts ........................... 68 5.2 Engineering for chain length control in vitro ............................................... 69 5.2.1 KS ....................................................................................................... 70 5.2.2 MPT .................................................................................................... 76 5.2.3 AT ....................................................................................................... 79 5.2.4 ACP domain-domain interactions ........................................................ 81 5.2.5 Interplay of domains and understanding network complexity .............. 84 5.3 Engineering beyond chain length control ................................................... 86 5.3.1 Engineering FAS for the production of a polyketide ............................ 86 5.3.1 Production of short FA in vivo using S. cerevisiae .............................. 87 5.4 Implications on other megasynthases ........................................................ 90 5.5 Outlook ....................................................................................................... 91 5.6 Conclusion ................................................................................................. 92 6 Scientific supplementary information ................................................................. 94 6.1 Supplementary Notes ................................................................................. 94 6.2 Supplementary Figures ............................................................................ 108 6.3 Supplementary Tables ............................................................................. 123 7 Statement of personal contributions ................................................................ 129 7.1 Scientific work .......................................................................................... 129 7.2 Writing process ......................................................................................... 131 8 Additional indexes ........................................................................................... 132 8.1 List of figures ............................................................................................ 132 8.2 List of tables ............................................................................................. 134 8.3 Abbreviations............................................................................................ 135 9 Literature ......................................................................................................... 137 10 Non-scientific supplementary ....................................................................... 152 10.1 Danksagung ............................................................................................. 152 10.2 Eidesstattliche Erklärung .......................................................................... 153 1 Deutsche Zusammenfassung 1.1 Hintergrund In der Natur kommen Fettsäuren (FA) in allen eukaryotischen und bakteriellen Organismen vor. Die für die Herstellung verantwortlichen Enzyme werden als Fettsäuresynthasen (FAS) bezeichnet1. In den letzten Jahren erhielt die Erforschung von FAS eine erneute Aufmerksamkeitssteigerung, da ihr Potential für biotechnologische Anwendungen erkannt wurde2: Die durch FAS hergestellten FA können eine Schlüsselrolle in größeren Stoffwechselwegen spielen und weiter zu Methyl-/Ethylestern, Aldehyden, Fettalkoholen oder Alkanen umgesetzt werden3. Diese wiederum finden Anwendung als Lebensmittelzusätze, Emulgatoren und auch als Bestandteil von Biokraftstoffen. Der
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