INVESTIGATING THE LONG-CHAIN POLYUNSATURATED FATTY ACID BIOSYNTHESIS OF THE AFRICAN CATFISH CLARIAS GARIEPINUS (BURCHELL, 1822) THESIS SUBMITTED TO THE UNIVERSITY OF STIRLING FOR THE DEGREE OF DOCTOR OF PHILOSOPHY by ANGELA O. OBOH FEBRUARY 2018 INSTITUTE OF AQUACULTURE, SCHOOL OF NATURAL SCIENCES, UNIVERSITY OF STIRLING, STIRLING, SCOTLAND, UK DECLARATION This thesis has been composed in its entirety by the candidate. Except where specifically acknowledged, the work described in this thesis has been conducted independently and and has not been submitted for any other degree. Name: Angela O. Oboh Sign: Date: Name: Óscar Monroig Sign: Date: Name: Douglas R. Tocher Sign: Date: 1 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisors Dr Óscar Monroig and Prof. Douglas R. Tocher for their guidance, encouragement and help throughout this project. I am especially indebted to Dr. Óscar Monroig, who taught me most of the experimental methodologies used in this work. I would like to thank Dr. Monica Betancor and Dr Naoki Kabeya who also helped me understand aspects of the laboratory work and for their contributions to the success of this project. I would like to express my appreciation to Dr Juan Carlos Navarro (Instituto de Acuicultura Torre de la Sal (CSIC), Spain) for his support in analysing the fatty acids of the yeast samples reported in Chapter 4 of this thesis. I am thankful to Prof. Brett Glencross for his time and support as well. I am grateful to the staff of the molecular and nutrition laboratories and the tropical aquarium including Dr John Taggart, Mrs Jacquie Ireland, Mr Keith Ransom, Mr. James Dick, Dr Matthew Sprague, Mrs Fiona Strachan, Mrs Elizabeth Mackinlay and Mrs Irene Younger for their advice, support and willingness to help in the laboratory. I am grateful to the Commonwealth Scholarship Commission for funding this PhD. I would like to thank all my friends and colleagues at Stirling, especially my office mates through the years, for their friendship and support, and for all the very interesting conversations. I am grateful to my parents, brothers, sisters and extended family members for their love, support and prayers throughout the duration of my studies. Finally, I give all the glory to God who saw me through to the end of this project and who never left me alone through this period of my life. 2 ABSTRACT Investigating the biosynthesis of long-chain (C20–24) polyunsaturated fatty acids (LC- PUFA), physiologically important compounds including arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in fish is crucial to identify dietary requirements for essential fatty acids (EFA). Moreover, knowledge of the C20–24 LC-PUFA biosynthetic capability of farmed fish species enables us to understand their ability to utilise commonly used raw materials such as vegetable oils, which naturally lack LC-PUFA but include C18 PUFA that are metabolic precursors of LC-PUFA. Studies have shown that the potential of a species for LC-PUFA biosynthesis is associated with the complement and function of fatty acyl desaturase (fads) and elongase of very long chain fatty acid (elovl) genes existing in that species. The present study therefore aimed to investigate these genes in the African catfish (Clarias gariepinus), the most commercially important farmed fish in sub-Saharan Africa. A fads2, a fads6 and four elovl (elovl2, elovl4a, elovl4b, elovl8) cDNAs were cloned and functionally characterised by heterologous expression in yeast. The Fads2 was a bifunctional desaturase enzyme with ∆6∆5 and ∆8 activities, and thus catalysing all the desaturation reactions required for ARA and EPA biosynthesis from C18 precursor fatty acids. Moreover, the C. gariepinus Fads2 enzymes also desaturated 24:5n-3 to 24:6n-3, a ∆6 desaturation required for the biosynthesis of DHA through the so-called “Sprecher pathway”. Functional characterisation of Fads6 by heterologous expression in yeast did not reveal its function. With regards to elongases, the C. gariepinus Elovl2 demonstrated the ability to elongate C20 and C22 PUFA and thus complements the Elovl5 with elongase capability towards C18 and C20 PUFA. The Elovl8 was capable of only limited elongation of C18 and C20 PUFA. Elovl4a and Elovl4b, enable the biosynthesis of very long-chain (>C24) fatty acids, compounds with major roles in vision and fertility of vertebrates. The present study confirmed that C. gariepinus possess all the enzymatic capabilities required for the biosynthesis of ARA, EPA and DHA and, therefore, its physiological EFA requirements could be satisfied with dietary provision of C18 PUFA. 3 TABLE OF CONTENTS DECLARATION ............................................................................................................. 1 ACKNOWLEDGEMENTS ............................................................................................. 2 ABSTRACT ..................................................................................................................... 3 TABLE OF CONTENTS ................................................................................................. 4 LIST OF ABBREVIATIONS .......................................................................................... 9 LIST OF FIGURES ....................................................................................................... 12 LIST OF TABLES ......................................................................................................... 15 CHAPTER 1. .................................................................................................................. 17 GENERAL INTRODUCTION ........................................................................................ 17 1.1 Current Status of Fish Production ........................................................................ 18 1.1.1 Production of the African Catfish, Clarias gariepinus ................................. 19 1.1.2 Clarias gariepinus Nutrition ......................................................................... 23 1.1.3 Lipid Sources and Essential Lipids for C. gariepinus Feed Production ....... 24 1.2 Fatty Acids: Classification and Nomenclature .................................................... 25 1.3 Fish Essential Fatty Acid Requirements .............................................................. 27 1.4 Biological Functions of Fatty Acids in Fish ........................................................ 32 1.5 Fatty Acid Synthesising Enzymes ....................................................................... 34 1.5.1 Fatty Acyl Desaturases ................................................................................. 34 1.5.2 The Desaturation of Fatty Acids ................................................................... 36 1.5.3 Classification and Activities of Fads enzymes ............................................. 37 1.5.4 Elongation of Very Long-chain Fatty Acid (Elovl) protein .......................... 39 1.5.5 Classification and Activities of Elongation of Very Long-chain Fatty acid (Elovl) Enzymes..................................................................................................... 40 1.5.6 Biosynthesis of Long-Chain Polyunsaturated Fatty Acids (LC-PUFA) in Fish ................................................................................................................................ 42 1.5.7 LC-PUFA Biosynthetic Capabilities of Clarias gariepinus ......................... 44 1.6 Objectives of This Study ...................................................................................... 45 CHAPTER 2. .................................................................................................................. 47 GENERAL MATERIALS AND METHODS ................................................................... 47 2.1 Materials .............................................................................................................. 48 2.2 Preparation of Media, Buffers and Gels .............................................................. 48 2.2.1 Preparation of 50x TRIS/acetate/EDTA (TAE) Buffer (500 ml) ................. 48 4 2.2.2 Preparation of Luria-Bertini (LB) Medium and Agar (400 ml) .................... 49 2.2.3 Preparation of Competent Escherichia coli Cells ......................................... 49 2.2.4 Preparation of Yeast Extract Peptone Dextrose (YPD) Medium and Agar (100 ml) .................................................................................................................. 50 2.2.5 Preparation of Competent Saccharomyces cerevisiae Cells ......................... 50 2.2.6 Preparation of Na Salts of Fatty Acids .......................................................... 51 2.2.7 Preparation of S. cerevisiae Minimal Medium (SCMM-ura) (400 ml) ........... 51 2.2.8 Preparation of S. cerevisiae Minimal Medium Plates (200 ml) .................... 52 2.3 Gene Molecular Cloning ...................................................................................... 52 2.3.1 Experimental Samples ................................................................................... 52 2.3.2 RNA Extraction ............................................................................................. 52 2.3.3 First Strand cDNA Synthesis......................................................................... 54 2.3.4 Amplification of cDNA Fragments ............................................................... 54 2.3.5 RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)