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Characterization and Engineering of Novel Sucrose/Trehalose Synthases

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Characterization and Engineering of Novel Sucrose/Trehalose Synthases Unlocking nature’s glycosylation potential: characterization and engineering of novel sucrose/trehalose synthases ir. Margo Diricks Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Academic year: 2016-2017 Promotor Prof. dr. Tom Desmet (Ghent University) Faculty of Bioscience engineering Department of Biochemical and Microbial Technology Centre for Synthetic Biology Coupure Links 653, 9000 Ghent – Belgium Dean Prof. dr. ir. Marc Van Meirvenne Rector Prof. dr. Anne De Paepe Examination committee Prof. dr. ir. Dirk Reheul (Ghent University, Chairman) Prof. dr. ir. Matthias D’hooghe (Ghent University, Secretary) Prof. dr. ir. Bartel Vanholme (Ghent University, VIB) Prof. dr. ir. Wim Van den Ende (KU Leuven) Prof. dr. ir. Yves Briers (Ghent University) Prof. dr. Tom Desmet (Ghent University) To refer to this thesis Diricks, M. (2017) Unlocking nature’s glycosylation potential: characterization and engineering of novel sucrose/trehalose synthases. PhD thesis, Faculty of Bioscience Engineering, Ghent University, Ghent. Acknowledgements This work was supported by the Special Research Fund (BOF, doctoral scholarship) of Ghent University as well as the European Commission FP7-project ‘SuSy’ (grant nr. 613633). Cover illustration Designed by Margo Diricks ISBN 978-94-6357-018-3 Copyright © 2017 by Margo Diricks. All rights reserved. The author and the promotor give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Table of contents List of abbreviations .............................................................................................................. 1 Introduction ........................................................................................................................... 3 CHAPTER 1: Literature review .............................................................................................. 9 1 Carbohydrate active enzymes ....................................................................................10 1.1 Introduction .........................................................................................................10 1.2 Classification ......................................................................................................11 1.3 Glycosyltransferases ..........................................................................................11 1.4 GT4 family ..........................................................................................................15 2 Sucrose Synthase ......................................................................................................16 2.1 Reaction, classification and mechanism .............................................................16 2.2 Substrate promiscuity .........................................................................................17 2.3 Structure .............................................................................................................17 2.4 Factors influencing expression and activity .........................................................19 2.5 Biotechnological applications ..............................................................................21 3 Construction of phylogenetic trees .............................................................................22 4 Enzyme engineering ..................................................................................................24 4.1 Engineering substrate specificity .........................................................................25 4.2 Engineering protein stability ................................................................................30 4.3 Engineering of GTs: case studies .......................................................................32 5 Supplementary materials ...........................................................................................35 CHAPTER 2: Identification and characterization of novel bacterial SuSy enzymes ..............39 1 Abstract .....................................................................................................................40 2 Introduction ................................................................................................................41 3 Materials and methods ...............................................................................................42 3.1 Materials .............................................................................................................42 3.2 Phylogenetic analysis .........................................................................................42 3.3 Cloning of novel SuSy genes in a constitutive expression system.......................45 3.4 Construction of truncation mutants of SuSyAc ....................................................47 3.5 Enzyme production and purification ....................................................................47 3.6 Enzyme assays...................................................................................................48 3.7 SDS-PAGE analysis ...........................................................................................50 3.8 Western blot analysis ..........................................................................................50 3.9 Effect of pH, temperature and divalent cations on SuSy activity ..........................51 3.10 Nucleotide sequence accession numbers ...........................................................51 3.11 Statistical analysis ..............................................................................................51 4 Results and discussion ..............................................................................................52 4.1 Phylogenetic and taxonomic analysis of prokaryotic SuSy sequences ................52 4.2 Recombinant expression of novel prokaryotic SuSys ..........................................54 4.3 Effect of pH, temperature and divalent cations on the activity of novel prokaryotic SuSys ...........................................................................................................................55 4.4 Kinetic properties and substrate specificity of novel prokaryotic SuSys ...............57 4.5 Production of UDP-glucose with SuSyAc ............................................................59 4.6 Truncation mutants of SuSyAc ............................................................................60 4.7 Genomic organization of SuSy genes in bacteria ................................................61 4.8 Phosphorylation of prokaryotic SuSys .................................................................64 5 Conclusions ...............................................................................................................65 6 Supplementary materials ...........................................................................................67 CHAPTER 3: Improving the affinity of a bacterial SuSy for the nucleotide acceptor UDP .....77 1 Abstract .....................................................................................................................78 2 Introduction ................................................................................................................79 3 Materials and methods ...............................................................................................79 3.1 Amino acid distribution ........................................................................................79 3.2 Site-directed mutagenesis ..................................................................................80 3.3 Enzyme production and purification ....................................................................81 3.4 Characterization of variant SuSys .......................................................................81 3.5 Coupled reactions ...............................................................................................81 3.6 Homology modeling ............................................................................................82 4 Results and discussion ..............................................................................................83 4.1 Nucleotide preference and its relation to the QN motif ........................................83 4.2 Mutational analysis of the QN motif .....................................................................85 4.3 Coupled reaction between SuSy and a C-glycosyltransferase ............................88 5 Conclusions ...............................................................................................................90 6 Supplementary materials ...........................................................................................91 CHAPTER 4: Driving the donor specificity of SuSy towards GalFru, for the efficient one-step production of UDP-galactose ................................................................................................97 1 Abstract .....................................................................................................................98 2 Introduction ................................................................................................................99 3 Materials and methods ............................................................................................. 100 3.1 Materials ..........................................................................................................
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