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Biotechnological production of galactosides of pharmaceutical interest: enzyme screening, engineering and application 90) - 02 - Estela Castilla Ypas http://hdl.handle.net/10803/565506 Generalitat 472 (28 de Catalunya núm. Rgtre. Fund. ió ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de undac la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials F d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs. Universitat Ramon Llull Universitat Ramon ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o G: 59069740 G: materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la . Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la C.I.F persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices. WARNING . The access to the contents of this doctoral thesis and its use must respect the rights of the author. It can be used for reference or private study, as well as research and learning activities or materials in the terms established by the 32nd article of the Spanish Consolidated Copyright Act (RDL 1/1996). Express and previous authorization of the author is required for any other uses. In any case, when using its content, full name of the author and title of the thesis must be clearly indicated. Reproduction or other forms of for profit use or public communication from outside TDX service is not allowed. Presentation of its content in a window or frame external to TDX (framing) is not authorized either. These rights affect both the content of the thesis and its abstracts and indexes. C. Claravall, 1-3 | 08022 Barcelona | Tel. 93 602 22 00 | Fax 93 602 22 49 | [email protected] | www.url.edu 90) - 02 - de Catalunya núm. 472 (28 472 núm. Catalunya de DOCTORAL THESIS Generalitat Generalitat Title Biotechnological production of galactosides of pharmaceutical interest: enzyme screening, engineering and application Rgtre. Fund. Rgtre. Fundació Fundació Presented by Estela Castilla Ypas Universitat Ramon Llull Llull Ramon Universitat Centre IQS School of Engineering G: 59069740 59069740 G: . Department Bioengineering C.I.F Directed by Dr. Antoni Planas and Dra. Teresa Pellicer C. Claravall, 1-3 | 08022 Barcelona | Tel. 93 602 22 00 | Fax 93 602 22 49 | [email protected] | www.url.edu Per tots aquells que ho han fet possible. I especialment per als meus pares. Eloi i Rosa, va per vosaltres. “…amar la trama más que el desenlace” 5 Acknowledgments Un cop s’arriba al final d’una etapa és important mirar enrere, fer balanç i recalcar- ne allò més important. Després d’aquests 4 anys de doctorat he après infinitat de coses i puc dir que he crescut com a científica i com a persona. Aquest apartat intenta agrair i recalcar tot allò que he guanyat en aquesta época. Primer m’agradaria agrair al Pla del Doctorat Industrial (Agaur i Generalitat de Catalunya) per atorgar-nos aquesta beca. Ha estat una fantàstica oportunitat per conèixer el món de la recerca a la universitat i compartir-ho amb la indústria. També vull agrair a IQS i a Interquim S.A haver-me permès moure’m entre els dos mons amb tanta facilitat i comoditat. Agrair al Dr. Antoni Planas, director d’aquesta tesi, confiar en mi i donar-me la possibilitat de realitzar aquest doctorat en el seu laboratori. Gràcies per les discussions i crítiques, és un plaer aprendre tanta ciència d’algú com tu. Gràcies per fer-me descobrir que el món dels carbohidrats pot donar més energia del que sembla! Agrair a la Dra. Teresa Pellicer, directora d’aquesta tesi, primer de tot per confiar en mi: tan en l‘inici com en el llarg de tota la tesi. Gràcies per tota la ciència que has compartit i per la teva manera tant instructiva d’ensenyar. També m’has ensenyat a veure que més enllà de la ciència, hi ha punts i comes a tindre en compte. Moltes gràcies per fer-me veure molt més que números i enzims! També vull agrair al Dr. Xavier Camps i al Dr. Francisco Marquillas permetre’m conèixer Interquim i el projecte gaxilosa des de ben aprop. Gràcies per confiar en mi i en la biocatàlisi. Gràcies per tant gran oportunitat. També vull donar les gràcies al grup del projecte gaxilosa amb els que he tingut la sort de treballar: ha estat un plaer treballar amb vosaltres i veure la investigació des dels ulls de la indústria. Part de lo aconseguit en aquest treball és vostre! Fede, gracias por estar siempre 100% disponible, sé que al final le has cogido cariño a las enzimas. Santos, gracias por tu ayuda, por ensenyarme la parte más industrial del proceso. He aprendido muchísimo en las visitas a los laboratorios de Interquim. Cristina, gràcies per la teva visió analítica: els teus anàlisis han fet palpable els èxits del projecte. També m’agradaria agrair a la Paqui totes les gestions. Moltes gràcies per fer-ho tot TAN fàcil! Thanks to Dr. Charles Tellier to allow me entering to your laboratory and work with you and your team. It was a wonderful opportunity and a great experience. Merci beaucoup pour tout! 6 Gracias, también, al Dr. Alberto Marina por darme la posibilidad de acercarme al complicado, pero apasionante, mundo de la cristalografía. Fue todo un reto para mí! Gracias por todos los resultados compartidos! També vull agrair al tribunal haver acceptat llegir i avaluar aquest treball. Espero que sigui una lectura agradable i en disfruteu tan com jo.// Thanks to the jury for accepting the evaluation of this work. I hope you a nice lecture and I hope you enjoy it as I much as I did. Donar les gràcies a la Dra. Magda Faijes. Podria dir que el doctorat va començar amb tu durant el master en bioenginyeria. Gràcies pel teu entusiasme per la ciència i les ganes de saber més. Durant el master vas transmetre les ganes d’apendre i descrobir que sempre tens, i ho has mantingut durant aquest 4 anys on hem compartit laboratori, seminaris i reunions. Encara recordo aquell dinar on, crec, vaig començar a buscar doctorats. Moltes gràcies! Gràcies al Dr. Xevi Biarnés. Agrair la teva entrega del 100% a bioquímica. Saps que això no seria el mateix sense tu i els teus ulls computacionals. Gràcies per compartir les teves idees i punts de vista. T‘agraeixo les aportacions que has fet en aquest treball. Al teu costat sempre se n’aprèn moltíssim!! Moltes gràcies! Agrair a diferents investigadors amb els que he pogut compartir hores al laboratori i aprendre, cada dia, de cada un de vosaltres: Amanda (gràcies per fer-nos començar el dia sempre amb un somriure), Dra. Marta Palomo (gràcies per demostrar que amb ganes, tot es pot), Dr. Xavi Pérez (gràcies per compartir tants coneixements d’anàlisi!), Dr. Edu Andrés (gracias por tus consejos en el mundo glicosintasa), Dr. Régis Fauré (thanks for you help during your stay, your desire to collaborate and share science. And thanks for the science you share with us, it was very helpful. Merci beaucoup!). Així com investigadors d’altres departaments com la Dr. Gemma Gotor i la Dra. Cristina Ribas, per ajudar sempre amb els problemes cromatogràfics! Als diferents professors i col·laboradors de bioquímica: Dr. Pau Leiva, Dr. Marc Carnicer i Patri. Han estat 3-4 anys amb vosaltres compartint seminaris, pica-pica, reunions i dinars. Ha estat un plaer treballar al vostre costat. Gràcies per la vostra ajuda i idees! He après molt de vosaltres! Que seria de quaselvol doctorat sense els cafés al bar de l’IQS... moltes gràcies a tot l’equip per fer d’aquests breaks una festa continua!! Per descomptat també agrair al personal d’IQS, recepció, reprografia, manteniment i magatzem, la disposició a solucionar imprevistos i resoldre embolics. Gràcies per fer el dia a dia més fàcil! I també més entretingut! 7 Durant aquests 4 anys he tingut la sort de compartir laboratori amb un munt gent, i de cada un d’ells me n’he quedat alguna cosa, amb o sense el seu permís. Cris (Dra. Val) fuiste un ejemplo a seguir, y demostraste que la maña siempre puede más que la fuerza.
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  • Building a Glycosylation Platform in E. Co/I Through Metabolic Engineering Frederik De Bruyn

    Building a Glycosylation Platform in E. Co/I Through Metabolic Engineering Frederik De Bruyn

    Building a Glycosylation Platform in E. co/i through Metabolic Engineering Frederik De Bruyn Examination committee Prof. dr. ir. Paul Van Der Meeren (Ghent University, Chair) Prof. dr. ir. Matthias D'hooghe (Ghent University) Prof. dr. Magda Faijes (Universitat Ramon Llull, Spain) Prof. dr. ir. Els Van Damme (Ghent University) dr. ir. Manu De Groeve (Ablynx) Prof. dr. ir. Wim Soetaert (Ghent University) Prof. dr. ir. Marjan De Mey (Ghent University) Supervisors Prof. dr. ir. Wim Soetaert (Ghent University) Prof. dr. ir. Marjan De Mey (Ghent University) Department of Biochemical and Microbial Technology, Center of Expertise – Industrial Biotechnology and Biocatalysis, Ghent University Dean Prof. dr. ir. Guido Van Huylenbroeck Rector Prof. dr. Anne De Paepe Ghent University Faculty of Bioscience Engineering Department of Biochemical and Microbial Technology Building a Glycosylation Platform in E. coli through Metabolic Engineering Frederik De Bruyn Thesis submitted for the fulfillment of the requirements For the degree of Doctor (PhD) in Applied Biological Sciences Academic year: 2014-2015 Dutch translation of the title: Ontwikkeling van een glycosyleringsplatform in E. coli door Metabolic Engineering To refer to this thesis: De Bruyn, F. (2014) Building a Glycosylation Platform in E. coli through Metabolic Engineering. PhD thesis, Faculty of Bioscience Engineering, Ghent University, Ghent. Cover illustration: ©iStockphoto.com Edited by Brecht De Paepe and Frederik De Bruyn ISBN 978-90-5989-744-1 Copyright © 2014 by Frederik De Bruyn. 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.
  • Milk Biosynthesis

    Milk Biosynthesis

    FairLife: Cold Filtration • https://www.youtube.com/watch?v=YgHFaNVofY8 Milk Biosynthesis Part 5: Lactose, Mineral, and Vitamin Secretion Lactose is… • The major carbohydrate in milk • Present in milk of most mammals • A readily digestible source of glucose for the neonate • Digested by lactase enzyme in neonate • Unique to the mammary gland • The major osmoregulator of milk (draws water into gland) Lactose Concentration • Least variable milk component • High lactose concentration will not exist • More lactose = more water coming into alveolar lumen keeping lactose concentration fairly constant • Low lactose concentration will occur during involution and some illnesses • Remember milk is the only source of water for a neonate so lactose is very important!! What is lactose? • Over 60% of whole body glucose use in the lactating dairy cow is for lactose synthesis • Lactose = 1 molecule glucose + 1 molecule galactose • Glucose alone is the primary substrate for lactose synthesis • One glucose is converted to UDP-glucose, which is converted to one UDP-galactose • Another glucose is used for lactose synthesis without modification • 2 glucoses are required for each lactose molecule synthesized Glucose • Glucose passes across the Golgi membrane into the Golgi lumen by a glucose transporter (GLUT 1) • Presence of GLUT 1 on the Golgi membrane is specific to the mammary epithelial cell • Glucose transport does not require energy, but it is affected by glucose levels in cytoplasm Lactose Synthesis • UDP-galactose is actively transported into the