ELUCIDATION OF THE BIOSYNTHETIC PRODUCTION PATHWAYS OF NEUTRAL LIPIDS IN THE MARINE HAPTOPHYTE EMILIANIA HUXLEYI ____________ A Thesis Presented to the Faculty of California State University, Chico ____________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology ____________ By Lindsey Kaylee Wallace Fall 2012 ELUCIDATION OF THE BIOSYNTHETIC PRODUCTION PATHWAYS OF NEUTRAL LIPIDS IN THE MARINE HAPTOPHYTE EMILIANIA HUXLEYI A Thesis by Lindsey Kaylee Wallace Fall 2012 APROVED BY THE DEAN OF GRADUATE STUDIES AND VICE PROVOST FOR RESEARCH: _________________________________ Eun K. Park, Ph.D. APROVED BY THE GRADUATE ADVISORY COMMITTEE: _________________________________ Gordon Wolfe, Ph.D., Committee Chair _________________________________ David Keller, Ph.D. _________________________________ Daniel Edwards, Ph.D. ACKNOWLEDGMENTS This work was supported by NSF grant EFRI-0938157. I would like to thank Mark Brown, from Iowa State University, for GC-MS analysis of PULCA components. Katie Scott for help with radio labeling experiments in the Summer of 2012. Thanks to Sarah Hoddick who provided positive feedback, immense emotional support, and death metal. I also owe appreciation to my committee members, Dr. Edwards and Dr. Keller, for their guidance and assistance that made my thesis possible. Most of all, thank you to Dr. Wolfe for not only the opportunity to work in his lab but also continued guidance and support throughout my project. iii TABLE OF CONTENTS PAGE Acknowledgments……………………………………………………………………. iii List of Tables………………………………………………………………………..... vi List of Figures……………………………………………………………………….... vii Abstract……………………………………………………………………………...... ix CHAPTER I. Introduction………………………………………………………………. 1 E. huxleyi: A Globally-Important Algae……………………………… 1 E. huxleyi Neutral Lipids……………………………………………... 1 PULCA Accumulation and Cell Stress………………………………. 5 Biofuel Applications…………………………………………………. 6 PULCA Localization…………………………………………………. 7 Possible Biosynthetic Pathways…………………………………….... 8 Study Objectives, Questions, and Hypothesis………………………... 12 II. Materials and Methods………………………………………………….... 15 Cultures……………………………………………………………….. 15 Nile Red Staining, Microscopy, and Fluorometry……………………. 16 Lipid Extractions……………………………………………………... 17 Thin Layer Chromatography…………………………………………. 18 Gas Chromatography – Mass Spectrometry Analysis………………... 18 iv CHAPTER PAGE Radioisotope Uptake Experiments…………………………………..... 20 III. Results…..……………………………………………………………….... 23 GC-MS Analysis of Neutral Lipids…………………………………… 23 Tracing Carbon Flow Into Neutral Lipids Over a Growth Cycle…...... 29 Carbon Flow Into Neutral Lipids Over Light-Dark Manipulations…... 34 Effect of Inhibitors on Carbon Flow Into Neutral Lipids………........... 40 IV. Discussion……………………………………………………………….... 45 Identification of Neutral Lipids with GC-MS………………………... 45 Changes in Production of Neutral Lipid Types Based on Growth Conditions……………………........................................................ 46 Acetate Utilization……………………………………………………. 47 Bicarbonate Utilization……………………………………………...... 48 Inhibitor Effects on Neutral Lipid Synthesis…………………………. 49 Summary of Findings…………………………………………………. 51 Possible PULCA and C31-33 cis-alkene Synthesis Pathways………….. 52 References…………………………………………………………………………........ 55 v LIST OF TABLES TABLE PAGE 1. Variations in LC alkenes in E. huxleyi strains showing presence of C31-33 cis-alkenes and/or C37-39 trans-alkenes…………………….. 4 2. Provasolli-Guillard Culture Collection (NCMP) algal cultures……….... 16 3. Inhibitors used in pulse radio labeling experiments…………………...... 20 4. Major hydrocarbon types identified in I. galbana (1323) and E. huxleyi strains using GC-MS analysis…………………………………….. 26 5. Comparison of bicarbonate vs. acetate uptake………………………….. 41 6. Summary of early inhibitor effects on 14C-acetate uptake and 14C-bicarbonate into CCMP 1516………………………………… 42 7. Summary of inhibitor effects on 14C-acetate uptake into CCMP 1516…. 43 8. Summary of inhibitor effects on 14C bicarbonate uptake into CCMP 1516 and 3268 ………………………………………………. 44 vi LIST OF FIGURES FIGURE PAGE 1. PULCA skeletons with diunsaturated trans bonds at w14,21............... 2 2. C31-33 LC alkene skeletons with cis geometry………………………… 3 3. Biosynthetic pathways proposed by Rontani et al. (2006)…………... 10 4. A model of polyunsaturated FA (PUFA) and PULCA biosynthesis ... 12 5. Diagram of lipid extraction procedure ………………………………. 22 6. Separation of nonpolar lipids using TLC and then identification of lipids with GC-MS……………………………………………….. 25 7. Growth of 1516 and 3268 over 14 days……………………………… 28 8. Pools of neutral lipids in exponential vs. stationary with bicarbonate E. huxleyi cells…………………………………………………..... 29 9. Cell growth of cultures for pulse chase experiment………………….. 31 10. Pulse chase radio labeling of E. huxleyi strain 1516 with 14C Acetate. 32 11. Pulse chase radio labeling of E. huxleyi strain 1516 with 14C bicarbonate…………………………………………………… 33 12. Light-dark experiment schematic……………………………………... 35 13. Neutral lipid per cell over light-dark cycle………………………........ 35 14. Neutral lipid pools per cell over light-dark cycle……………….......... 37 15. Light effects 14C bicarbonate uptake……………………………......... 39 vii FIGURE PAGE 16. 14C acetate pulse labeling in the dark, and testing for bacterial controls............................................................................................ 40 17. TLC plate of hydrocarbon, neutral lipid, and PULCA extraction method............................................................................................. 42 viii ABSTRACT ELUCIDATION OF THE BIOSYNTHETIC PRODUCTION PATHWAYS OF NEUTRAL LIPIDS IN THE MARINE HAPTOPHYTE EMILIANIA HUXLEYI by Lindsey Kaylee Wallace Master of Science in Biology California State University, Chico Fall 2012 Emiliania huxleyi and some related prymnesiophyte algae produce a novel group of polyunsaturated long-chain C37-39 alkenones, alkenoates, and alkenes as their major neutral lipids, however their biosynthesis pathway is unknown. Like triglycerides, these lipids are believed to be utilized as storage lipids and are accumulated in lipid bodies to be used as a fuel source, presumably. Also C31-33 cis-alkenes have been identified in E. huxleyi but are believed to be different in synthesis and function. By studying the synthesis of these lipids I set out to discern how these two types of neutral lipids are formed in E. huxleyi. I used a combination of techniques, including GC-MS analysis, radiolabeling, and inhibitors, to examine lipid pools during growth cycles, bicarbonate dosing, and light-dark manipulations. By using GC-MS analysis I identified ix the presence of C31-33 cis-alkenes exclusively in E. huxleyi strains CCMP 1516 and 371, and Isochrysis galbania strain CCMP 1323; where E. huxleyi strains CCMP 1742, 3266, and 3268 had both cis-alkenes and C37-38 trans-alkenes. I also found accumulation of both cis-alkenes and C37-38 trans-alkenes in light to dark manipulations was similar to that seen in storage lipids, suggesting that the long chain cis-alkenes are in fact another storage lipid and may share a similar synthesis pathway to the trans-alkenes. Using radiolabeling studies I found external acetate is not acquired under light dependent mechanisms and is utilized primarily in production of polar lipids; while external bicarbonate acquisition and use in lipid synthesis is light-dependent, and as a cellular building block it is distributed more evenly amongst lipid pools. Also, flow of carbon into CCMP 1516 and 3268 cells from external bicarbonate into lipid pools is inhibited by cerulenin (fatty acid synthase inhibitor), flufenacet (elongase inhibitor), and quizaloflop (acetyl-CoA carboxylase inhibitor). Platensimycin (fatty acid synthase II inhibitor) only affects flow of bicarbonate into CCMP 1516 C31-33 cis-alkenes. Finally, flow of carbon into CCMP 1516 cells from external acetate into lipid pools is inhibited by cerulenin. Flufenacet and quizaloflop only affects flow of acetate into C37-39 trans-alkenes. Platensimycin has no effect on the flow of acetate into lipid pools in CCMP 1516. Though these results show precursors of acetate and bicarbonate are used in the synthesis of these neutral lipids, and through inhibitor studies I have identified many mechanisms vital to their synthesis, the biosynthetic pathway is yet unclear. x CHAPTER I INTRODUCTION Emiliania huxleyi: A Globally-Important Algae Emiliania huxleyi is a marine haptophyte algae, found ubiquitously in oceans. This single celled phytoplankton is typically 3-7 µm in diameter and has a triphasic life cycle. The complex life cycle of E. huxleyi involves several stages with distinct cell types: the coccolith-bearing diploid motile cells, vegetative diploid naked cells, and haploid motile scale-bearing swarm cells; each stage can exist independently and reproduce (Laguna et al 2001, Rokitta et al 2011). They form a large part of marine biomass and are the most abundant of the coccolithophorids. Coccolithophorids are organisms that produce coccoliths, which are calcium carbonate disks that are extruded to the surface of the cell, their function is still unknown. E. huxleyi can accumulate in large white blooms observable from space as dense as 107 cells per L (Olson & Strom 2002), the white color is due to the shedding of coccoliths produced by the cells. E. huxleyi’s ability to produce coccoliths and abundance makes them an important part of the biological carbon cycle and contributor to atmospheric composition