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Characterization and Engineering of Epimerases for the Production of Rare Sugars

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Characterization and Engineering of Epimerases for the Production of Rare Sugars Characterization and engineering of epimerases for the production of rare sugars Koen Beerens Members of the jury: Prof. dr. ir. Wim Soetaert (promotor) Prof. dr. Tom Desmet (promotor) Prof. dr. Els Van Damme (secretary) Prof. dr. ir. Chris Stevens (chairman) Em. Prof. dr. ir. Erick Vandamme Em. Prof. dr. Matti Leisola dr. Jo Maertens Promotors: Prof. dr. ir. Wim Soetaert Prof. dr. Tom Desmet Centre of Expertise - Industrial Biotechnology and Biocatalysis (InBio.be) Department of Biochemical and Microbial Technology Ghent University, Belgium Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Paul Van Cauwenberge Koen Beerens was supported by a SB-fellowship (SB81309 and SB83309) of the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen). The research was conducted at the Centre of Expertise - Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University (Ghent, Belgium). Koen Beerens CHARACTERIZATION AND ENGINEERING OF EPIMERASES FOR THE PRODUCTION OF RARE SUGARS Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences (Cell and gene biotechnology) Dutch translation of the title: Karakterisering en engineering van epimerasen voor de productie van zeldzame koolhydraten To refer to this thesis: Beerens, K. (2013) . Characterization and engineering of epimerases for the production of rare sugars. PhD thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, 198 p Printed by University Press, Zelzate ISBN-number: 978-90-5989-588-1 Copyright © 04/02/2013 by Koen Beerens. All rights reserved. The author and the promotors 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. Dankwoord De laatste weken heb ik ettelijke keren volgende of een gelijkaardige zin zien staan in de verschillende vacatures die ik bekeken heb: “Je kan zelfstandig werken, maar je bent ook een echte teamplayer”. Deze zin, hoe alledaags en triviaal die ook mag klinken, is misschien wel de kortste samenvatting die je van een doctoraatsonderzoek kan maken. Een doctoraat is immers een persoonlijk project maar wel eentje dat je niet kan realiseren zonder de steun en hulp van vrienden, familie en collega’s. Bij deze wil ik dan ook iedereen bedanken die me de voorbije vier jaar heeft gesteund en geholpen. Eerst en vooral wil ik mijn promotoren Prof. dr. ir. Wim Soetaert en Prof. dr. Tom Desmet bedanken om vijf jaar geleden in een pasafgestudeerde biochemicus te geloven en zo mijn eerste werkervaring te geven. Maar ook voor de kans om nadien mijn eigen doctoraatsonderzoek te starten en mij hier het nodige vertrouwen en geduld voor te geven. Wim, jou wil ik nog bedanken om me destijds een andere kant van onderzoek te laten zien, namelijk het toegepaste i.p.v. het fundamentele onderzoek alsook om me de wondere wereld van suikers en de kristallisatie hiervan te laten ontdekken. Tom, voor jou een woordje van dank voor alle hulp bij mijn beursaanvraag, het schrijven van mijn artikels, de bemoedigende woorden in ontmoedigende tijden, me de kans te geven om in Cambridge over mijn onderzoek te gaan spreken, maar uiteraard ook voor minder wetenschappelijke momenten op congressen. De volledige “Glycodirect”-groep wil ik bedanken voor de leuke sfeer op het bureau en het labo, maar ook voor de wetenschappelijke discussies. Laten we maar beginnen bij de intussen ex- collega’s. Manu, bedankt om me in te leiden in de enzyme engineering alsook in de automatisatie hiervan. Ook voor Eefje een dankuwel voor de hulp en uitleg bij InBios robotica. An, veel succes bij Ablynx en dat je er op de volgende InBio-(after)party wel mag geraken. Giang, let’s just thank you for being a €5-communist, your giggly smile and the delicious Vietnamese snacks. Chen Chao, Chen or Chao, I will never forget the amazing “Yes, but …” moment. Jef, zonder jou is de humor een stuk minder droog op de Glycodirect-bureau. Dirk, merci voor de vijf leuke jaren. Barbara, een superdikke merci voor al jouw hulp met de ontwikkeling van mijn screeningsassay en het andere werk op de epimerasen. En dat jullie nieuwe spruit je niet te veel slapeloze nachten bezorgt zodat je die cellulasen kan bedwingen. Karel, met jou in de buurt is er altijd veel plezier te beleven (en schunnige praat te horen). Tom, merci om op de goede (en soms minder gepaste) momenten de boel op te vrolijken met “Statler en Waldorf”-gewijze opmerkingen en de grapjes tot ver buiten de Glycodirect-bureau. De nieuwkomers Magali en Renfei zou ik vooral veel succes, geduld en doorzettingsvermogen willen toewensen. Daarnaast wil ik ook mijn twee thesisstudenten, Simon en Koen, bedanken voor de hulp bij het praktische werk van mijn onderzoek, ook al heb ik door jullie mijn eerste grijze haren gekregen. Iedereen veel succes met jullie onderzoek en/of verdere carrière en dat we elkaar zeker nog ergens mogen tegenkomen! Verder wil ik natuurlijk ook alle andere (ex-)collega’s bedanken voor de leuke jaren samen op Limab/InBio. Emeritus Prof. dr. ir. Erick Vandamme, bedankt om voor mij jouw toga en wetenschappelijke kennis nog eens boven te halen. De mensen van het secretariaat voor de hulp met al het papierwerk. De geweldige gazet Liesbet, succes met je studies. Barbara, bedankt voor het last-minute nalezen van mijn doctoraat. Alle laboranten, doctoraatsstudenten en post-docs zowel voor hun hulp bij onderzoeksgerelateerde zaken als voor de geweldige sfeer die er altijd is geweest op InBio (en hopelijk nog lang mag blijven). Wouter aka dj Wally om steeds weer voor de ambiance te zorgen. Eric, merci voor de oppeppende gesprekken en de (zondag)avonden in ’t Spijker en de Hot Club de Gand. Kwok, Maarten, Catherine, Inge en Sophie voor de verticale ontspanning (lees: klimmuur en bijpassende Gulden Draak). Lien, wat een afscheidsfeestje om nadien gewoon weer terug te keren. Pieter, Gert, Frederik en Robin, dat er meer ups dan downs mogen zijn in jullie onderzoek de komende jaren. Sophie, veel succes met de laatste loodjes van jouw doctoraat! Marjan, supervrouw hier en in Amerika. Joeri, iemand die zowat alles weet staan en kent op InBio, merci. Jo, bedankt voor de leuke discussies en om je door mijn doctoraat te wurmen. Veel succes met jullie spin-off. Sofie, voor de hulp bij moleculair werk en de grappige momenten in het moleculair labo. En alle andere (ex-)InBio’tjes, bedankt voor alles! En natuurlijk de spelers en supporters van de InBio- en FBW-voetbalploegen niet vergeten bedanken voor zowel de sportieve als vloeibare ontspanning en uiteraard voor de kampioenschapstitel. Als laatste wil ik ook mijn ouders, familie en vrienden bedanken voor alles wat jullie voor mij betekenen en gedaan hebben. Moeke en vake, een dikke knuffel voor jullie onvoorwaardelijke steun. Dries, meestal ver weg maar er valt altijd op je te rekenen. Jan en Marhaba, merci voor de steun in moeilijke tijden. Jelle, Sten, Flip, Ruben, Huub alsook de andere mannen van “den OLK” en “De Mart” om op tijd en stond de batterijen helemaal te ledigen en weer op te laden. Charlotte, bedankt om in de laatste weken van mijn doctoraat voor ontspanning en steun te zorgen. Table of contents I. INTRODUCTION ............................................................................................. 1 II. LITERATURE REVIEW ................................................................................. 7 1 BIOCATALYTIC PRODUCTION ROUTES FOR RARE SUGARS ............................. 9 1.1. Rare sugars and their applications .................................................... 9 1.2. Enzymes for rare sugar production .................................................. 12 1.2.1. Oxidoreductases ................................................................................. 17 1.2.2. Isomerases.......................................................................................... 18 1.2.2.1. L-arabinose isomerase for tagatose production ............................... 19 1.2.2.2. Isomerases for L-ribose production ................................................ 20 1.2.2.3. Isomerases for deoxygenated and other modified sugars ............... 20 1.2.3. Carbohydrate epimerases ................................................................... 20 1.2.3.1. Ketohexose 3-epimerase ................................................................. 21 1.2.3.2. UDP-Galactose 4-epimerase .......................................................... 21 1.2.3.3. Cellobiose 2-epimerase .................................................................. 22 1.2.3.4. L-ribulose-5-phosphate 4-epimerase and related aldolases ............. 22 1.3. Rare sugars: conclusions and outlook.............................................. 23 2 THE RARE SUGAR TARGET: D-TAGATOSE .................................................... 24 2.1. Introduction ...................................................................................... 24 2.2. Properties ......................................................................................... 24 2.3. Production methods .......................................................................... 26 2.3.1. Chemical ............................................................................................ 26 2.3.2. Biochemical ....................................................................................... 27 2.4. Applications .....................................................................................
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