RECOMBINANT PRODUCTION OF OMEGA-3 FATTY ACIDS IN E. COL1 USING A GENE CLUSTER ISOLATED FROM SHEWANELLA BALTICA A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph By MITRA AMIRI-JAMI In partial fulfillment of requirements For the degree of Doctor of Philosophy January, 2009 © Mitra Amiri-Jami, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-58261 -9 Our file Notre reference ISBN: 978-0-494-58261-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada ABSTRACT RECOMBINANT PRODUCTION OF OMEGA-3 FATTY ACIDS IN E. COLI USING A GENE CLUSTER ISOLATED FROM SHEWANELLA BALTICA Mitra Amiri-Jami Advisor: University of Guelph, 2009 Professor M. W. Griffiths Omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play an important role in many aspects of human health. The main dietary source of EPA and DHA is cold-water fish, the populations of which are in decline. There has been extensive research to find alternative sources for EPA/DHA. Production of EPA and DHA by some bacterial strains such as Shewanella has been of interest for the purpose of cloning omega-3 fatty acid genes and recombinant production of EPA/DHA in other microorganism or higher organism such as plants. The first study presented in this thesis demonstrates the production of EPA/DHA in Shewanella baltica strains, which have been previously reported as non-omega-3 fatty acid-producing bacteria. All Shewanella baltica strains produced EPA and Shewanella baltica MAC1 synthesized both EPA and DHA. Next, we cloned EPA/DHA genes from Shewanella baltica MAC1 by constructing a bacterial artificial chromosome (BAC) library. Recombinant production of EPA/DHA was detected in one clone with a DNA insert size of 200 kb. Attempts to subclone the EPA/DHA gene cluster into a food grade, broad host range vector were not successful due to an inability to digest and to isolate the insert from the BAC vector. In chapter four, first each pfa (pfaA, pfaB, pfaC, pfaD, pfaE) gene of Shewanella baltica MAC1 responsible for EPA/DHA production was partially amplified in order to make probes for rapid detection of positive clones in a fosmid library. Second, to clone the EPA/DHA gene cluster, we prepared a fosmid library from Shewanella baltica MAC1 with insert size of -40 kb. Fosmid clones were screened by colony hybridization for pfaA and pfaD genes. Recombinant production of EPA was detected in six fosmid clones and one clone produced both EPA and DHA. The insert carrying EPA/DHA genes was isolated from the fosmid DNA using Noil. The research presented in this thesis demonstrated recombinant production of omega-3 fatty acid in E. coli and provided a DNA fragment (35 kb) containing all EPA/DHA genes which can be ligated to a food grade, broad host range vector in order to provide alternative sources of EPA and DHA. ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Mansel Griffiths who gave me the opportunity to work in his lab and for his trust that I was the right person for this project. He has provided advice, support, patience and respect while allowing me to maintain my independence, and for that, I am indebted and grateful. I would like to thank the members of my advisory committee. Dr. Yukio Kakuda who has always been available for long scientific discussions, support, encouragements and his expert knowledge. Dr. George Van der Merwe for his insightful comments and attention to detail. I would like to thank the many friends that I have made over the years for their support and memories that I will cherish forever. I would like to thank my parents who have always encouraged me to pursue my dreams. I would also like to thank my sisters and my brother for the bond we share. You have been my confidant, my supporter and my best friends. And to my family, I cannot say enough. My husband, Ali, who has loved and supported me no matter what. His patience, encouragement, understanding and scientific advice have been greatly appreciated. He made the rough times more bearable and the good times even better. To our two sons, Amin and Nima, I am in awe of their wisdom, patience and unbelievable understanding. You two have been a blessing to us and look forward to watching you grow. I love you and your father very much and I could not be a person who I am today without you. Finally, I thank God, the most compassionate, the most merciful, who makes all things possible. Table of Contents 1. General Introduction 1 1.1 An introduction to omega-3 fatty acids 1 1.2 The importance of omega-3 fatty acids 2 1.2.1 Omega-3 fatty acids and cardiovascular disease 4 1.2.2 Omega-3 fatty acids and cancer 6 1.2.3 Omega-3 fatty acids and diabetes 8 1.3 Sources of omega-3 fatty acid and recommended dietary intakes for the omega-3 fatty acids 9 1.4 An Introduction to Shewanella Species 10 1.5 The genus Shewanella and omega-3 fatty acid production 12 1.6 Mechanism of polyunsaturated fatty acid biosynthesis in bacteria 13 1.7 Genetic Studies in Shewanella Species 17 1.8 Methods for cloning the omega-3 fatty acid genes 20 1.9 Rationale 26 1.10 Research objectives 28 2. Reclassification of Shewanella putrefaciens MAC1 and detection of EPA/DHA production by Shewanell baltica strains 29 2.1 Abstract 29 2.2 Introduction 30 2.3 Materials and Methods 33 2.3.1 Bacterial strains 33 2.3.2 Preparation of Shewanella baltica cultures 34 2.3.3 Growth conditions and biochemical characteristics 34 2.3.4 DNA extraction from Shewanella baltica strains 35 ii 2.3.5 Polymerase Chain Reaction (PCR) procedure for 16S rRNA sequence determination 36 2.3.6 Separation of DNA fragments 36 2.3.7 Purification of amplified DNA fragment from PCR reactions 38 2.3.8 Growth conditions for fatty acid extraction 38 2.3.9 Extraction of fatty acids 38 2.3.10 Gas Chromatographic Analysis 39 2.3.11 Gas Chromatographic-Mass Spectrometry Analysis 40 2.3.12 Statistical Analysis 42 2.4 Results and discussion 42 2.4.1 Physiological properties and phenotypic characteristics of S. baltica strains 2.4.2 16S rRNA sequence analysis 42 2.4.3 Fatty acid composition of Shewanella baltica strains at different temperatures 45 2.4.4 EPA production by Shewanella baltica strains at different temperatures .... 48 2.5 Conclusions 56 3. Cloning omega-3 fatty acid genes from Shewanella balticaMACl by constructing a Bacterial Artificial Chromosomelibrary 57 3.1 Abstract 57 3.2 Introduction 58 3.3 Material and Methods 61 3.3.1 Bacterial strains and growth conditions 61 3.3.2 Genomic DNA isolation 62 3.3.2.1 DNA extraction using commercial kits 62 3.3.2.2 Extraction of DNA in plug agarose 63 3.3.3. Partial digestion of isolated chromosomal DNA with kits and in plugs 63 3.3.4 Pulsed Field Gel Electrophoresis 65 3.3.5 DNA recovery from the gel slices 65 3.3.5.1 Recovering of digested DNA from the gel slices using Gelase 65 iii 3.3.5.2 Recovering of DNA from gel slices by Electroelution 66 3.3.6. Construction of Bacterial Artificial Chromosome (BAC) Libraries 67 3.3.6.1 Ligation of DNA fragments into the CopyControl pCCBAC Cloning- Ready_Vector 67 3.3.6.2 Transformation, Plating and Selecting the BAC Clones 69 3.3.6.3 Sizing the BAC clones and storage of the BAC DNA 69 3.3.7 Screening of the Library 71 3.3.7.1 Growth condition 71 3.3.7.2 Fatty acid extraction 71 3.3.7.3 Gas Chromatographic Analysis 71 3.3.8 Analysis of EPA/DHA positive clones 72 3.3.8.1 Bacterial strain and growth conditions 72 3.3.8.2 BAC DNA isolation from positive clones 73 3.3.8.3 Complete digestion of BAC DNA 73 3.3.9 Determination of EPA/DHA production by positive clones in Skim milk..