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DEVELOPMENT of CRISPR/CAS9 in NANNOCHLOROPSIS and OTHER ALGAE TOWARD UNDERSTANDING and MANIPULATING ENERGY ALLOCATION by Brian V

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DEVELOPMENT of CRISPR/CAS9 in NANNOCHLOROPSIS and OTHER ALGAE TOWARD UNDERSTANDING and MANIPULATING ENERGY ALLOCATION by Brian V DEVELOPMENT OF CRISPR/CAS9 IN NANNOCHLOROPSIS AND OTHER ALGAE TOWARD UNDERSTANDING AND MANIPULATING ENERGY ALLOCATION by Brian Vogler A thesis submitted to the Faculty and the Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Applied Chemistry). Golden, Colorado Date _______________________ Signed: ___________________________ Brian W. Vogler Signed: ___________________________ Dr. Matthew C. Posewitz Thesis Advisor Golden, Colorado Date _______________________ Signed: ___________________________ Dr. Thomas Gennett Department Head Department of Chemistry ii ABSTRACT Nannochloropsis is a genus of eukaryotic microalgae that grows well in outdoor bioreactors and produces high yields of triacylglycerols (TAGs), which can be processed into biodiesel. In this work, we chemically characterize the storage carbohydrate of Nannochloropsis for the first time, and interrupt enzymes required for its biosynthesis to (1) understand their function and (2) interrogate whether this unused biomass component can be eliminated without significant impact on viability. To generate targeted gene knockouts, we developed CRISPR/Cas9 methods for Nannochloropsis and first interrupted nitrate reductase, a common target for genetic knockout because it is both inessential and easily scored. The method and validated chassis strain was then used to interrogate the beta-glucan synthase (BGS) and transglycosylase (TGS) enzymes believed to be responsible for the backbone polymerization and branching of the storage beta glucan, respectively. We identified no significant growth defects in our laboratory culturing conditions but did confirm that both genes were fundamental to synthesis of this beta glucan storage carbohydrate. The generated knockouts of either gene do not produce the elevated carbohydrate phenotype of wild-type cells in response to nitrogen deprivation. We also observed a non-bleaching phenotype in knockouts of BGS, where chlorophyll and carotenoid content remain elevated in mature cultures when wild-type cultures reduce their chlorophyll and carotenoid content. The design and diagnostic CRISPR/Cas9 methods developed for Nannochloropsis were then modified to transform a fast-growing high-light-, high-heat-, and high-salt-tolerant microalga Chlorella sp. strain CCMP252 with complexed Cas9-sgRNA to generate insertional knockouts of the nitrate reductase gene, demonstrating the broad applicability of the lessons learned. iii TABLE OF CONTENTS ABSTRACT ................................................................................................................................ iii LIST OF FIGURES .................................................................................................................... ix LIST OF TABLES ......................................................................................................................xiii ACKNOWLEDGEMENTS ........................................................................................................ xiv CHAPTER 1 INTRODUCTION .................................................................................................... 1 1.1 Photosynthetic microorganisms control the global environment ....................................... 1 1.2 Eukaryotic algae .............................................................................................................. 3 1.3 Nannochloropsis as a new model system ........................................................................ 5 1.4 Intracellular accumulation of fats and carbohydrates ....................................................... 6 1.5 Algal cell walls ................................................................................................................. 8 1.6 Mixotrophic and heterotrophic cultivation ....................................................................... 12 1.7 Focus of this work.......................................................................................................... 13 CHAPTER 2 CHARACTERIZATION OF PLANT CARBON SUBSTRATE UTILIZATION BY AUXENOCHLORELLA PROTOTHECOIDES ................................................................ 14 2.1 Introduction.................................................................................................................... 14 2.2 Materials and methods .................................................................................................. 15 2.2.1. Cultivation ....................................................................................................... 15 2.2.2. Metabolite 1D 1H-NMR .................................................................................... 17 2.2.3. FAME analysis ................................................................................................ 17 2.2.4. Glycome profiling ............................................................................................ 18 2.2.5. Proteomic analysis .......................................................................................... 18 2.2.5.1. Computerized comparisons ........................................................... 19 2.2.5.2. MW and pI measurements ............................................................. 19 2.2.5.3 Protein digestion and peptide extraction ........................................ 20 2.2.5.4. NanoLC-MS/MS ............................................................................ 20 iv 2.2.5.5. Data processing and protein identification ..................................... 21 2.2.6. Genomic sequencing, assembly, and annotation ............................................ 22 2.2.7. Transcriptomic sequencing and analysis ......................................................... 24 2.3. Results and discussion .................................................................................................. 25 2.3.1. Degradation of cellulose and plant substrates ................................................. 25 2.3.2. Genome analysis ............................................................................................ 31 2.3.3. Proteomics and transcriptomics ...................................................................... 33 2.4. Conclusions ................................................................................................................... 40 2.5. Acknowledgements ....................................................................................................... 41 CHAPTER 3 CHARACTERIZATION OF THE NANNOCHLOROPSIS GADITANA STORAGE CARBOHYDRATE: A 1,3-BETA GLUCAN WITH LIMITED 1,6-BRANCHING ............... 42 3.1. Introduction.................................................................................................................... 43 3.2. Materials and methods .................................................................................................. 44 3.2.1. Carbohydrate extraction .................................................................................. 44 3.2.2. 1H-NMR analysis ............................................................................................. 45 3.2.3. Size exclusion chromatography ...................................................................... 45 3.2.4. Linkage analysis ............................................................................................. 46 3.2.5. Degrees of polymerization and branching calculations .................................... 46 3.2.6. Phylogenetic analysis...................................................................................... 47 3.3. Results and discussion .................................................................................................. 47 3.3.1. Nannochloropsis produces a β-1,3-linked soluble glucan with β-1,6 branching ........................................................................................................ 47 3.3.2. Glucan synthase and branching enzyme are conserved among stramenopiles ................................................................................................. 50 3.3.3. Nannochloropsis may utilize a glycogenin-like protein to nucleate laminarin ... 51 3.3.4. Bioinformatics suggests three potential laminarinases from N. gaditana ......... 52 3.3.5. Manipulating carbohydrate synthesis increases lipid accumulation in diatoms ........................................................................................................... 54 3.4. Conclusions ................................................................................................................... 56 v 3.5. Acknowledgement ......................................................................................................... 56 CHAPTER 4 CAS9-GENERATED GLUCAN SYNTHASE KNOCKOUT OF NANNOCHLOROPSIS GADITANA ............................................................................... 57 4.1. Introduction.................................................................................................................... 57 4.1.1. Nannochloropsis storage carbohydrate ........................................................... 57 4.1.2. CRISPR/Cas9 nuclease-directed insertional mutagenesis .............................. 58 4.2. Materials and methods .................................................................................................. 59 4.2.1. Design and assembly of Cas9 expression vector for Nannochloropsis gaditana .......................................................................................................... 59 4.2.2.
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