I UNIVERSITY of CALIFORNIA, SAN DIEGO Insights Into Mechanisms Of
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Insights into mechanisms of high productivity and cultivation flexibility resulting from physiological and omic analyses of the microalga Marinichlorella kaistiae KAS603 A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Marine Biology by Eva Luz Sánchez Alvarez Committee in charge: Mark Hildebrand, Chair William Gerwick Brian Palenik Julian Schroeder José Luis Stephano Hornedo Victor Vacquier 2017 i Copyright Eva Luz Sánchez Alvarez, 2017 All rights reserved ii The Dissertation of Eva Luz Sánchez Alvarez is approved, and it is acceptable in quality and form for publication on microfilm and electronically: __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ Chair University of California, San Diego 2017 DEDICATION This dissertation is dedicated to my son, Guillermo Salomón Sánchez Alvarez, for your love, for being you, and for putting up with my crazy ideas and mood swings. I truly admire the wonderful person you have become and I am sure that only great things await you. With love and admiration from your mom. iv EPIGRAPH “Sometimes the questions are complicated and the answers are simple.” Dr. Seuss v TABLE OF CONTENTS Signature Page ........................................................................................................ iii Dedication ............................................................................................................... iv Epigraph .................................................................................................................. v Table of Contents ................................................................................................... vi List of Tables ......................................................................................................... vii List of Figures ....................................................................................................... viii Acknowledgements ................................................................................................ xi Vita ....................................................................................................................... xiii Abstract of the Dissertation .................................................................................. xiv Introduction ............................................................................................................. 1 Chapter 1. Evaluating Marinichlorella kaistiae KAS603 cell size variation, growth and TAG accumulation resulting from rapid adaptation to highly diverse trophic and salinity cultivation regimes. ................................................................. 7 Chapter 2. Genome sequence determination for characterization of metabolic processes in Marinichlorella kaistiae KAS603 ..................................................... 21 Chapter 3. Transcriptomic analysis of genes involved in multiple fission and carbon metabolism in Marinichlorella kaistiae KAS603 ................................... 116 vi LIST OF TABLES Table 1-1: Summary of M. kaistiae KAS603 top 4 cultivation conditions ........... 16 Table 2-1: Mk KAS603 list of genes from mitochondrial DNA .......................... 34 Table 2-2: Mk KAS603 list of genes from chloroplast DNA ................................ 38 Table 2-3: Mk KAS603 gene/protein assembly comparison of AUGUSTUS and FGENESH ............................................................................................................ 43 Table 2-4: Mk KAS603 protein localization prediction for enzymes involved in starch metabolism, glucose anabolism and catabolism ....................................... 55 Table 2-5: Mk KAS603 protein localization prediction for enzymes involved in the pyruvate hub ................................................................................................... 56 Table 2-6: Mk KAS603 Intracellular targeting predictions for enzymes that act in fatty acid and TAG anabolism ............................................................................. 57 Table S2-1: Abbreviations for enzymes corresponding to starch metabolism, glycolysis, gluconeogenesis, sucrose metabolism and pyruvate hub, in Mk KAS603 .............................................................................................................. 112 Table S2-2: Abbreviations for enzymes corresponding to fatty acid and TAG anabolism, in Mk KAS603 ................................................................................. 113 Table S2-3: Abbreviations for enzymes corresponding to the glyoxylate cycle, in Mk KAS603 ........................................................................................................ 114 Table S2-4: Abbreviations for enzymes corresponding to fatty acid and TAG catabolism, in Mk KAS603 ................................................................................. 115 Table 3-1: Summary of Mk KAS603 cultivation conditions used for gene expression comparison ....................................................................................... 133 vii LIST OF FIGURES Figure 1-1: Marinichlorella kaistiae KAS603 response to different molar concentations of bicarbonate in freshwater ............................................................. 9 Figure 1-2: M. kaistiae KAS603 response to different molar concentrations of acetate in freshwater cultures ................................................................................ 10 Figure 1-3: Cell area population distribution comparison under bicarbonate and acetate cultivation in freshwater ............................................................................ 11 Figure 1-4: Growth adaptation process of M. kaistiae KAS603 to increasing salinities ................................................................................................................. 12 Figure 1-5: Response comparison of salinity adapted M. kaistiae KAS603 in three NaCl concentrations .................................................................................... 13 Figure 1-6: Seawater mixotrophic vs autotrophic cultures of M. kaistiae KAS603 ............................................................................................................................... 14 Figure 1-7: Cell area population distribution comparison among cells cultured in ASW with low (A), medium (B) and high (C) NaCl amounts during day 9 of cultivation .............................................................................................................. 15 Figure 1-8: Outdoor cultures of M. kaistiae KAS603 at a pilot plant .................. 16 Figure 1-9: Proposed steps involved in bicarbonate (left) and acetate (right) uptake and utilization ........................................................................................... 17 Figure 1-10: Proposed processes involved in dealing with hypersaline conditions ............................................................................................................................... 18 Figure 2-1: Electrophoresis gel run for Mk KAS603 DNA purified from CsCl/Hoechst 33258 dye gradients ....................................................................... 31 Figure 2-2: Plastid genome arrangement among three Chlorella strains .............. 37 Figure 2-3: Genome size estimation of Mk KAS603 determined by flow cytometry ............................................................................................................... 40 viii Figure 2-4: Mk KAS603 correlation of gene mass and length of contigs ............ 44 Figure 2-5: Number of genes vs genome size in Mk KAS6003 ........................... 46 Figure 2-6: Genome comparison among 3 members of the family Chlorellacea: Marinichlorella kaistiae KAS603, Auxenochlorella protothecoides and Chlorella variabilis NC64A ................................................................................................. 49 Figure 2-7: Mk KAS603 predicted glycolysis, glucoenogenesis, starch metabolism and pyruvate hub topology map ........................................................ 51 Figure 2-8: Plastid amino terminal extension alignments for starch synthesis, glycolysis, gluconeogenesis and pyruvate hub enzymes ....................................... 61 Figure 2-9: Mk KAS603 predicted glyoxylate cycle topology map .................... 63 Figure 2-10: Mk KAS603 predicted TAG catabolism and FA beta oxidation topology map ......................................................................................................... 65 Figure 2-11: Mk KAS603 predicted FA-TAG synthesis and Acetyl CoA formation topology map ........................................................................................ 68 Figure 2-12: Mk KAS603 predicted photorespiration topology map .................. 71 Figure 2-13: Mk KAS603 predicted pentose phosphate pathway topology map .... ............................................................................................................................... 74 Figure 2-14: Topology map comparison of predicted localization of enzymes involved in starch metabolism, glycolysis, gluconeogenesis and the pyruvate hub in six green