Biosynthesis and Mobilization of Arachidonic-Acid-Rich Triacylglycerols in the Green Microalga Parietochloris Incisa Pushkar
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Biosynthesis and mobilization of arachidonic-acid-rich triacylglycerols in the green microalga Parietochloris incisa Thesis submitted in partial fulfillment of the requirements for the degree of "DOCTOR OF PHYLOSOPHY" by Pushkar Shrestha Submitted to the Senate of Ben-Gurion University of the Negev חשון תשס"ו 11/2005 Biosynthesis and mobilization of arachidonic-acid-rich triacylglycerols in the green microalga Parietochloris incisa Thesis submitted in partial fulfillment of the requirements for the degree of "DOCTOR OF PHYLOSOPHY" by Pushkar Shrestha Submitted to the Senate of Ben-Gurion University of the Negev Approved by the advisors: Prof. Zvi Hacohen ________________________ Prof. Bezalel Kessler ________________________ Dr. Inna Khozin-Goldberg ________________________ חשון תשס"ו 11/2005 Beer-Sheva TABLE OF CONTENTS Acknowledgments I Summary II List of figures and tables V List of abbreviations and symbols IX 1. INTRODUCTION 1 1.1. Polyunsaturated fatty acids 1 1.2. Importance of PUFAs 2 1.3. Occurrence of PUFA-rich TAG 6 1.4. Biosynthesis of TAG 7 1.4.1. De novo synthesis of FA 8 1.4.2. Chloroplastic and extrachloroplastic lipids 10 1.4.3. Membrane desaturases 12 1.4.4. Biosynthesis of C20 PUFA in algae 13 1.4.5. Partitioning of FA to TAG 16 1.4.6. Acyltransferases of TAG biosynthetic pathway 23 1.5. Factors affecting the biosynthesis of TAG 25 1.5.1. Temperature 25 1.5.2. Light 26 1.5.3. Nutrient deprivation 27 1.6. Mutant studies 30 1.7. Role of TAG 31 1.8. Working hypothesis 32 2. MATERIALS AND METHODS 34 2.1. Growth conditions 34 2.2. Growth Parameters 35 2.3. Lipid analysis 35 2.3.1. Lipid extraction 35 2.3.2. Fatty acid analysis 36 2.3.3. Lipid separation 37 2.3.4. Molecular species separation 38 2.3.5. Positional analysis of fatty acids distribution in individual lipids 39 2.4. Nitrogen starvation and recovery experiments 40 2.5. Inhibitor studies 40 2.5.1. Salicylhydroxamic acid (SHAM) 40 2.5.2. Sethoxydim 41 2.6. Characterization of TAG biosynthetic enzymes 41 2.6.1. Grinding and homogenization 41 2.6.2. Cellular fractionation 41 2.6.3. Enzymatic assays 42 2.7. Mutagenesis 44 3. RESULTS 45 3.1. Lipid studies 45 3.1.1. Lipid classes and fatty acid composition 45 3.1.2. Molecular species composition of PC 48 3.1.3. Molecular species composition of PE 50 3.1.4. Molecular species composition of DGTS 52 3.1.5. Molecular species composition of TAG 53 3.2. Role of extrachloroplastic lipids in TAG synthesis 56 3.2.1. Sethoxidim treatment 56 3.2.2. Phosphate starvation 58 3.2.3. P- and N-starvation 63 3.3. Enzymes of TAG biosynthesis 64 3.3.1. Optimization of cell homogenisation 64 3.3.2. Development of a protocol for cellular fractionation 67 3.3.3. DAGAT assay utilizing [1_14C]oleoyl-CoA and non-labeled DAG 71 3.3.4. DAGAT activity in microsomes 74 3.3.4.1. Effect of time and protein 74 3.3.4.2. Substrate specificity 74 3.3.4.3. Solubility and availability of DAG substrate in assay 75 3.3.4.4. Effect of thiol reagents 77 3.3.4.5. Effect of cations 78 3.3.4.6. The origin of labeled DAG and the evidence for the MAGAT activity 79 3.3.5. DAGAT assay in microsomes with [1_14C]DAG and non-labeled oleoyl-CoA 80 3.3.5.1. Protein and time dependence 81 3.3.5.2. Effect of oleoyl-CoA concentrations 83 3.3.5.3. Selectivity of acyl-CoA substrates 84 3.3.5.4. Effect of ethanol 85 3.3.5.5. Effect of thiol-reagent PCMB 85 3.3.5.6. Effect of salts 86 3.3.6. Acyl-CoA independent TAG synthesis from [1_14C]1,2-dioleoyglycerol 88 3.3.6.1. Time and protein dependence 88 3.3.6.2. pH and temperature dependence 89 3.3.6.3. Effect of thiol reagents PCMB, DTT and CuCl2 90 3.3.6.4. Distribution of activity in the cellular fractions 91 3.3.7. DAGAT activity in oil bodies in assay with [1-14C]oleoyl-CoA 92 3.3.8. DAGAT assay in oil bodies with [1-14C]dioleoylglycerol 94 3.3.8.1. Factors affecting DAGAT activity in oil bodies 95 3.3.9. MAGAT activity in microsomes 100 3.3.9.1. Effect of MAG concentration 101 3.3.9.2. Time dependence 101 3.3.9.3. Effect of ethanol 102 3.3.9.4. Effect of dioleoylglycerol addition 105 3.3.9.5. Effect of MgCl2 105 3.3.10. Lipolytic activity in microsomes 107 3.3.10.1. pH and temperature 107 3.3.10.2. Time and protein 110 3.3.10.3. Effects of divalent metals and inhibitors on DAG lipase activity 111 3.3.10.4. Positional specificity 112 3.3.11. Evidence for the activity of TAG lipase 113 3.4. Role of AA-rich TAG 114 3.4.1. Nitrogen starvation and recovery 114 3.4.1.1. Nitrogen starvation 114 3.4.1.2. Recovery from the nitrogen starvation 115 3.4.1.3. Alterations in lipid and fatty acid content and composition 117 3.4.1.4. Molecular species analysis 119 3.4.1.5. Radiolabelling 121 3.4.2. SHAM treatment and recovery 123 3.5. Mutant studies 128 4. DISCUSSION 132 4.1. Lipids involved in the biosynthesis of AA-rich TAG 132 4.2. Role of extrachloroplastic lipids in TAG synthesis 136 4.3. Enzymes of TAG biosynthesis 138 4.4. Role of TAG 150 4.4.1. Nitrogen starvation and recovery 150 4.4.2. Recovery from SHAM treatment 155 4.5. Mutant studies 156 5. REFERENCES 158 6. HEBREW ABSTRACT ACKNOWLEDGMENTS I would like to express my deep gratitude to my supervisors Prof. Zvi HaCohen, Prof. Bezalel Kessler and Dr. Inna Khozin-Goldberg for providing me an excellent opportunity to have this study and for their kind supervision and support to accomplish this dissertation research. I am very thankful to Inna for her sincere guidance in laboratory and in everyday life during the whole tenure in Israel, without which this study wouldn’t have been materialized. I am grateful to Shosh Didi-Cohen, Ilana Saller, Dorit Levin, Ben Friehoff and Amos Masika for their crucial help during this study and thanks to all my friends whom I met in Midreshet Ben Gurion for their kindness. I also would like to thank my teachers, Prof. Sanu Devi Joshi, Prof. Govinda Prasad Ghimire, Dr. Micha Guy, Dr. Micha Volokita and friends, Dr. Deepak Khadka and Dr. Roshan Shrestha, whose goodwill promoted me to commence this endeavor. I am highly indebted to my parents and aunt, who bestowed me with selfless love and care and continuous encouragement for study since my childhood. Thanks for their blessings. Finally, my heartfelt thank goes to my wife, Shiru and son, Sakar for their patience, understanding and every support in good and hard moments in Sde Boker. Her support, encouragement and companionship greatly facilitated my journey through this study. I SUMMARY The very long chain-polyunsaturated fatty acid (VLC-PUFA), arachidonic acid (AA, 20:4ω6) is essential for brain development and critical biological functions of human health. Over 30% of dry weight of the green microalga Parietochloris incisa are triacylglycerol (TAG) and over 95% of cellular AA are deposited in these TAG. The accumulation of AA-rich TAG brought about a great interest in exploring the mechanism of its biosynthesis and distinctive role in this alga. Analysis of lipid classes revealed that AA and its precursors are concentrated mostly in the extraplastidic lipids. The molecular species and stereo-specific analyses of fatty acid distribution of phosphatidylcholine (PC) revealed the presence of various C18 PUFAs and AA in the sn-2 and 16:0 in the sn-1 position and indicated that ∆12 and ∆6 desaturations occurred at the sn-2 position. Molecular species of phosphatidylethanolamine (PE), containing AA at the sn-1 position and C20 PUFA at the sn-2 position are likely to be involved in the ∆5 desaturation of 20:3. Diarachidonoyl-PC and -PE were among the major molecular species in both the exponential and stationary phases. The presence of the intermediates, C18:1-3 and C20:3, primarily in the sn-2 position of diacylglycerol (N,N,N)-trimethylhomoserine (DGTS), suggests the role of this position in the ∆12, ∆6 and possibly also in ∆5 desaturations. Most molecular species of TAG contain 2 or 3 AA moieties. We assume that AA synthesized in PE is exported to PC, DGTS and TAG and partly to the chloroplastic lipids. The AA/AA molecular species of PC and PE may have a key role in donating AA for TAG by either providing AA moieties to the acyl-CoA pool via the acyltransferases of the Kennedy pathway, or directly as DAG (diacylglycerol). AA derived from the sn-2 position of PE (18:1ω7/AA) can be also incorporated into the sn-1 and sn-3 positions of TAG. The significance of extraplastidic polar lipids in AA-rich TAG biosynthesis was studied following administration of sethoxidim, an inhibitor of the de novo fatty acids synthesis, and under P-starvation. II In order to characterize the final and committed step of TAG biosynthesis involving diacylglycerol acyltransferase (DAGAT), a cellular fractionation protocol was developed. This protocol enabled to minimize TAG degradation and ensure the isolation of intact oil bodies. When membrane fractions and oil bodies were applied in in vitro assays containing labeled substrates, [14C]oleoyl-DAG or [14C]oleoyl-CoA and unlabelled acyl-CoA or DAG, respectively, labeled TAG was formed, indicating the activity of DAGAT.