University of Groningen. In principio erat Lactococcus lactis Coelho Pinto, Joao Paulo
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1 University of Groningen In principio erat Lactococcus lactis Coelho Pinto, Joao Paulo IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Coelho Pinto, J. P. (2015). In principio erat Lactococcus lactis: Towards a membrane protein overproducer host ([Groningen] ed.) University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:
2 In principio erat Lactococcus lactis: Towards a membrane protein overproducer host João Paulo Coelho Pinto
3 Printing: Printed by Ipskamp Drukkers B.V., Enschede, the Netherlands Cover: Photo and cover design by João Pinto BTW: Latitude: ; Longitude: ISBN (Printed Version): ISBN (Digital Version): Printing of this thesis is financially supported by the Graduate School of Science and the University of Groningen. The work described in this thesis was carried out in the Molecular Genetics Group of the Groningen Biomolecular and Biotechnology Institute (Faculty of Mathematics and Natural Sciences, University of Groningen, The Netherlands)
4 In principio erat Lactococcus lactis Towards a membrane protein overproducer host PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Friday 29 May 2015 at hours By João Paulo Coelho Pinto born on 9 February 1979 in Faro, Portugal
5 Supervisors Prof. J. Kok Prof. B. Poolman Assessment committee Prof. A.J.M. Driessen Prof. J.M. van Dijl Prof. E.J. Smid
6 Dedicated to love, friendship, affection, solidarity... So many words, yet only one, Susana
7
8 CONTENTS Abstract Chapter 1 General Introduction Chapter 2 The Response of Lactococcus lactis to Membrane Protein Production Chapter 3 Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR Chapter 4 On the architecture of the CesSR regulon of Lactococcus lactis Chapter 5 ix The Growth-Dependent Transcriptome of Lactococcus lactis MG1363: Foundations for a Bacterial Biography Chapter 6 Upgrading the Toolkit of Lactococcus lactis: Part I: pseudo, a genetic integration standard for Lactococcus lactis Part II: Genomic Array Footprinting in Lactococcus lactis Chapter 7 Concluding Remarks Addendum (Extended) Summary for the layperson Resumo para leigos Samenvatting voor de leek Science & Society Acknowledgements List of Publications Brief Biography Contacts of Co-Authors vii
9 viii
10 ABSTRACT
11 Abstract Membrane proteins comprise an important class of proteins. They are involved in many essential processes, such as importing and secreting molecules, sensing of environmental parameters and scaffolding of cell structure. Their importance is easily affirmed as membrane proteins represent ~1/4 of all types of proteins of any organism and >60% of all drug targets. However, the study of membrane proteins is typically limited by inherent constraints, such as their hydrophobicity and added requirements for correct folding. These characteristics limit e.g. their production in appropriate quantities in a native form. We describe in this thesis a set of works that collectively aim at establishing Lactococcus lactis as a possible cellular factory for the production of membrane proteins. The physiological response of L. lactis to the overproduction of several membrane proteins was determined employing proteome and transcriptome methodologies. The data from these studies indicate that the two-component system CesSR, which senses cell envelope stresses from different origins, is one of the major response players when L. lactis is induced to overproduce membrane proteins. We also showed that overexpressing cessr improves the ability of L. lactis to produce membrane proteins for example the human protein PS1Δ9, which is linked to the susceptibility to Alzheimer, by more than 4 fold. We also describe several contributions to the genetic toolkit of L. lactis. In addition, we present gene expression data with unprecedented time resolution throughout the entire growth curve under standard laboratorial growth conditions of this widely used model organism. These contributions should benefit the entire lactococcal scientific community in answering many research questions that are still unresolved. x
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