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Engineering the Metabolism of Starmerella Bombicola for the Production of Tailor-Made Glycolipids

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Engineering the Metabolism of Starmerella Bombicola for the Production of Tailor-Made Glycolipids Ghent University Faculty of Bioscience Engineering Department of Biochemical and Microbial Technology Engineering the metabolism of Starmerella bombicola for the production of tailor-made glycolipids ir. Robin Geys Thesis submitted for the fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Academic year: 2016-2017 Examination committee: Prof. dr. ir. Mieke Uyttendaele (Ghent University, Belgium) Prof. dr. ir. Monica Höfte (Ghent University, Belgium) Prof. dr. Vlada Urlacher (Heinrich-Heine Universität, Germany) Dr. ir. Manu De Groeve (Ablynx NV, Belgium) Dr. ir. Sophie Roelants (Ghent University, Belgium) Promotors: Prof. dr. ir. Wim Soetaert (promotor) Prof. dr. ir. Inge Van Bogaert (co-promotor) Centre of Expertise-Biotechnology and Biocatalysis Department of Biochemical and Microbial Technology Ghent University Dean: Prof. dr. ir. Marc Van Meirvenne Rector: Prof. dr. Anne De Paepe Robin Geys was supported by Flanders Innovation & Entrepreneurship, formerly known as the institute Innovation by Science and Technology. Grant number 121129. Titel van het doctoraatsproefschrift in het Nederlands: Engineering van het metabolisme van Starmerella bombicola voor de productie van nieuwe glycolipiden. Cover illustration: Shutterstock ID 256289095 To refer to this thesis: Geys, R. (2016) Engineering the metabolism of Starmerella bombicola for the production of tailor-made glycolipids. PhD-thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. ISBN-number: 978-90-5989-968-1 The author and the promotors give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to copy right laws. Permission to reproduce any material contained in this work should be obtained from the author. List of Abbreviations 5-FOA 5-fluoroorotic acid ACP Acyl carrier protein AMP Adenosine monophosphate AOC Allene oxide cyclase AOS Allene oxide synthase APGs Alkyl polyglucosides AT Acetyltransferase ARS Autonomously replicating sequence bp Base pair CBL Cellobiose lipid CMC Critical micelle concentration CPR Cytochrome P450 reductase CTAB Cetrimonium bromide DES Divinyl ether synthase dTDP Deoxythymidine diphosphate EDTA Ethylenediaminetetraacetic acid ELSD Evaporative light scattering detector ER Endoplasmatic reticulum FAD Flavin adenine dinucleotide FAMEs Fatty acid methyl esters FAS Fatty acid synthase FMN Flavin mononucleotide GDP Guanosine diphosphate GFP Green fluorescent protein HAA 3-(3-hydroxyalkanoyloxy)alkanoic acids HPL Hydroperoxide lyase HPLC High-performance liquid chromatography HSL Homoserine lactone kb Kilobases LE Lactone esterase LB Luria-Bertani medium LC-MS Liquid chromatography–mass spectrometry MDR Multi-drug resistant ABC transporter MEL Mannosylerythritol lipid MEOR Microbial enhanced oil recovery MFE-2 Multifunctional enzyme type 2 MIC Minimum inhibitory concentration MRM Liquid chromatography–multiple reaction monitoring NaCl Sodium chloride NADH Reduced Nicotinamide adenine dinucleotide NADPH Reduced Nicotinamide adenine dinucleotide phosphate nat Nourseothricin acetyltransferase gene NDP Nucleoside diphosphate Ni-NTA Nickel-Nitrilotriacetic acid affinity resin NRPS Nonribosomal peptides synthetases P450 Cytochrome P450 monooxygenase PBS Phosphate buffer saline solution PCR Polymerase chain reaction pGAPD Glyceraldehyde 3’-phosphate dehydrogenase promotor pGKI Phosphoglycerate kinase promotor PGM Phosphoglucomutase PHA Polyhydroxyalkanoate PQS Pseudomonas quinolone signal QS Quorum sensing RhlA Rhamnosyltransferase chain A RhlB Rhamnosyltransferase chain B RhlC Rhamnosyltransferase chain C RmlA Glucose-1-phosphate thymidylyltransferase RmlB dTDP-D-glucose 4,6-dehydratase RmlC dTDP-4-keto-6-deoxy-d-glucose 3,5-epimerase RmlD dTDP-4-keto-L-rhamnose reductase RPLC Reverse phase liquid chromatography RPM Rotations per minute SD Synthetic dextrose medium SDS Sodium dodecyl sulphate TLC Thin layer chromatography UDP Uridine diphosphate UGTA1 UDP-glucosyltransferase A1 UGTB1 UDP-glucosyltransferase B1 ura3 Orotidine-5'-phosphate decarboxylase YPD Yeast peptone dextrose medium Acknowledgments “Moest je opnieuw kunnen kiezen, zou je het dan nog doen?” Dit is waarschijnlijk één van de meest gestelde vragen aan een doctoraatsstudent gedurende de 4 zware jaren van onderzoek. Velen zullen zeggen, zeker tijdens de beruchte 2-jaarsdip, dat ze het niet weten of zullen volmondig “nee” antwoorden. De mensen die me kennen kunnen het antwoord waarschijnlijk al wel raden. Voor zij die me niet zo goed kennen, het is een resolute “ja”. Hoewel er soms periodes waren waarin het onderzoek eerder leek op een beerkar die vastgereden was is een zompige akker kan ik niet ontkennen dat alle goede, mooie, leuke en vooral interessante momenten veruit de mindere overschaduwen. Natuurlijk heb ik dit niet alleen kunnen doen, vele mensen hebben me geholpen, bijgestaan of soms het bloed onder mijn nagels weten te krijgen in de aanloop naar dit doctoraat. Allereerst wil ik graag mijn ouders bedanken. Zonder hen zou ik hier nu niet staan met een vers doctoraat in de hand. Zij hebben me de afgelopen jaren altijd gesteund, me het volste vertrouwen gegeven en vooral altijd de vrijheid gegeven om te doen wat ik graag wou doen. Soms zullen ze zich wel afgevraagd hebben waarom ik godsnaam bepaalde zaken heb gedaan (of niet gedaan) maar zoals het goede ouders betaamt lieten ze de ruimte om zelf fouten te maken om je dan bij te staan indien nodig. In het huiselijke nest waren niet enkel mijn ouders een steun maar ook mijn zus had haar bijdrage. Iedereen met een broer of zus kan het wel beamen dat er gewoon eens zijn, eens onnozel doen of zelfs ruzie maken net die momenten zijn die blijven hangen. Hoewel familie je stuurt en steunt doorheen je leven zijn er speciale momenten waarop je mensen leert kennen die evengoed een grote impact hebben. Tijdens de lagere school en alle jaren erna zijn er twee personen die ik speciaal wil vermelden, Ian en Tim. Merci gasten voor de vele namiddagen, avonden en nachten. Verder denk ik ook aan het Sint-Michielscollege. Hoewel het niet direct de favoriete school van mijn ouders was ben ik hier toch begonnen aan mijn middelbare carrière. Spreken over een carrière is misschien vreemd voor de eerste drie jaar, de term is echter wel van toepassing op de laatste vier. Hoewel ik vele mensen heb leren kennen in die periode zijn er slechts enkelen die ik af en toe nog eens hoor. De eerste namen die me zo te binnen schieten zijn Laura, Toon, Michael, Damien maar nog vele meer verdienen aandacht. Naast mijn medestudenten hebben ook enkele leerkrachten me weten te “sturen” tot waar ik hier nu sta. Merci Cuypers en Merciny. Het meeste hoor ik echter nog van mevrouw Bwarompy, Markske a.k.a. Marcia Bwarody. Altijd klaar voor een voos nieuw avontuur tijdens een nachtwandeling, altijd paraat om te babbelen of te luisteren (naar mijn soms non-stop geratel) en vooral altijd bereidt om haar ongezouten mening te geven. Merci Marcia voor de ontelbare keren dat ik jouw mening nodig had (en vooral kreeg) over soms iets pietluttigs zoals welke koptelefoon zal ik kopen . Naast het middelbaar zijn de hogere studies een andere tijdsperiode waarin veel nieuwe ervaringen worden opgedaan. De start van mijn loopbaan aldaar was in de Biomedische Wetenschappen. Het moet gezegd worden dat deze start eerder een soort valse start was aangezien een 18-tal maanden later ik ingeschreven was als aspirant bachelor in de bio- ingenieurswetenschappen. Het aantal mensen dat ik heb leren kennen in die zes jaar is ontelbaar. Gelukkig is het aantal vrienden wel makkelijker te overschouwen. Ze allemaal opnoemen zou te lang duren dus een kleine selectie dient zich aan. Vier jaar heb ik doorgebracht op het studentenhome van campus Middelheim van de Universiteit Antwerpen. Hoewel ze eerder een rem op de studies te noemen waren wil ik toch mijn buren en kotgenoten vermelden. Merci Wouter, Karen, Ruben, Bart, Sam (zowel Puls als Verboven), Yannick, Alfons, Eef, Jens, Tim en Caroline voor de prettige avonden in de Konijnenpijp, Hagar, Buis, Profke, op de gang of op iemands kot. Daarnaast moet een student ook studeren. Hoewel bijna iedereen in de richting me pas heeft leren kennen tijdens de Zuid-Afrikareis (halverwege jaar 2) kan ik terugblikken op enkele mooie tijden met mijn studiegenoten. Vele van hen zie ik nog regelmatig en zijn waarschijnlijk ook aanwezig tijdens de verdediging. Merci Sven, Emilie, Elke, Laurent, Margo, Anouck en Daan. Ik ga me nu even verantwoorden, degene die langer in Gent zijn blijven hangen komen later. In ieder geval, een deel van de studie is uiteraard de masterthesis. Dit was mijn eerste echte aanraking met degelijk wetenschappelijk onderzoek (de amateurastronomie daarbuiten gelaten). Het is tijdens deze periode dat het zaadje van het doctoraat zich pas wist te planten. Hoewel de argumenten om me binnen te halen bij Inbio tijdens de thesis slechts deels vervuld zijn geraakt (je maakt enkele nieuwe giststammen; je zal dan fermenteren met deze stammen; je kan dan naar de piloot gaan als alles goed gaat; je bent welkom op de barbecue op de piloot) blik ik terug op een bewogen jaar met enkele maanden verplicht niet pipetteren. De sfeer, het onderzoek en de mensen waren ruimschoots voldoende om me te overtuigen om nog enkele jaren te blijven hangen. Gelukkig was ik niet alleen in Gent. Enkele studiegenoten zijn ook blijven plakken voor enkele jaren. Met hun kon ik regelmatig eens op restaurant of gewoon bij iemand gezellig gaan koken. Magali, Sanne, Timothy,
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