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Dissertation Investigating the Biochemistry and Genetics DISSERTATION INVESTIGATING THE BIOCHEMISTRY AND GENETICS OF CHRYSOLAMINARIN METABOLISM IN A MODEL MARINE DIATOM Submitted by Michael Adan Caballero Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2017 Doctoral Committee: Advisor: Graham Peers John Belisle Daniel Bush Ashok Prasad Copyright by Michael Adan Caballero 2017 All Rights Reserved ABSTRACT INVESTIGATING THE BIOCHEMISTRY AND GENETICS OF CHRYSOLAMINARIN METABOLISM IN A MODEL MARINE DIATOM Diatoms are ecologically significant marine algae as they contribute approximately 20 % of Earth’s photosynthetic productivity. The evolutionary history of diatoms is complex, and their chimeric genomes encode unexpected biochemistries. Diatoms are proposed to be an excellent candidate to produce sustainable biofuels and/or bioplastics; however, a limited functional understanding of their cellular biochemistries represents a critical challenge that limits the use of diatoms in industrial settings. Notably, diatoms make the unusual storage sugar chrysolaminarin, instead of glycogen or starch. The carbon stored in this carbohydrate pool may be exploited and redirected towards products of interest, such as the accumulation of triacylglycerol as a precursor metabolite for biofuel production. Optimizing carbon flow to biofuel or bioplastic precursors requires a fundamental understanding of how chrysolaminarin is made, modified, and consumed. Therefore, I have designed and executed a three-part research strategy to investigate the biochemistry and genetics of chrysolaminarin metabolism in the model diatom Phaeodactylum tricornutum. This research addresses key challenges in chrysolaminarin biology, including quantification of the polysaccharide, elucidating the first enzymatic step of chrysolaminarin biosynthesis, and identification of novel proteins associated with the chrysolaminarin metabolism. First, I set out to improve the state of the art of chrysolaminarin quantification, and this work is documented in Chapter 2. The 3-methyl-2-benzothiazolinone hydrazone (MBTH) assay ii affords considerably better accuracy than the phenol-sulfuric acid assay[1, 2]. This assay, when combined with a warm water extraction, permits parallel quantification of soluble non-reducing carbohydrates (chrysolaminarin), soluble reducing carbohydrates, and insoluble carbohydrates. This method was applied to an investigation of carbon partitioning in Phaeodactylum during nitrogen starvation, a treatment that has been shown to increase chrysolaminarin content in other diatoms[3]. Interestingly, Phaeodacylum did not increase chrysolaminarin content relative to total organic carbon during nitrogen starvation, challenging the assumption that diatoms universally repartition their carbon into chrysolaminarin reserves during nitrogen starvation. The outcome of this research was an accurate and reliable method to quantify carbohydrate fractions, especially chrysolaminarin, from diatom extracts. Second, I set out to systematically characterize diatom enzymes capable of synthesizing UDP-glucose, which is documented in Chapter 3. The E. coli strain DEV6 lacks a functional UDP-glucose diphosphorylase, conferring an inability to grow when galactose is the sole carbon source[4]. This deficiency has been used as a complementation assay to demonstrate the ability of a recombinant, eukaryotic protein to catalyze the synthesis of UDP-glucose [5]. I have adapted this strategy to facilitate a functional screen of a Phaeodactylum cDNA Expression Library. In this manner, I have identified a novel Phaeodactylum enzyme capable of synthesizing UDP- glucose, increasing the number of potential enzymes involved in the first step of chrysolaminarin biosynthesis to three. All three gene targets (ugp1 , ugp2 , and ugp3 ) were knocked out using a CRISPR/Cas9 approach and were quantified for chrysolaminarin content. Additionally, UDP- glucose can play other unique roles in the metabolism of photoautotrophs, such as sulfolipid biosynthesis[6], which was further explored. This research systematically explored the potential contribution of three UDP-glucose synthesizing enzymes to chrysolaminarin biosynthesis. The iii comprehensive suite of CRISPR/Cas9 knockouts revealed unique phenotypic patterns associated for each disrupted gene. Knocking out ugp1 confers mutants which accumulate less chrysolaminarin and more triacylglycerol at dusk. Knocking out ugp2 yields mutants that accumulate not only less chrysolaminarin but also less triacylglycerol at dusk. These ugp2 knockouts also exhibit reduced fitness both in exponential growth rates and maximum quantum yield of Photosystem II. Knocking out ugp3 does not yield an obvious phenotype relative to WT for the carbon partitioning or fitness parameters examined. The ugp1 knockouts exhibit phenotypes in good agreement with previous reverse genetics studies[7, 8]. These findings help resolve and predict distinct metabolic roles for these three enzymes, a critical detail when interpreting systems biology datasets or establishing genome-wide metabolic models. Third, I set out to identify novel, b -1,3 glucan binding proteins from the Phaeodactylum proteome, and these efforts are documented in Chapter 4. I implemented a 2D-affinity electrophoresis approach to resolve proteins based on their interaction with an embedded substrate. This strategy has resolved several starch binding proteins[9]. I adapted this approach by substituting b -1,3 glucan in the gel, and separating Phaeodactylum soluble protein extracts. A gel spot was resolved in this manner, and unique peptides were identified from the hypothetical protein Phatrdraft_47612. RNAi-mediated knockdowns of Phatrdraft_47612 were generated to investigate a potential chrysolaminarin phenotype. However, later analyses of protein migration after 2D-affinity electrophoresis suggested that the gel spot I observed might be an artifact of protein overloading. While this research effort did not meet the goal of identifying novel chrysolaminarin-related proteins, I have documented several considerations for future efforts to identify b -1,3 glucan-binding proteins by reflecting on shortcomings in the execution of this study. iv The following research chapters in this thesis aim to expand our understanding of chrysolaminarin metabolism in the model diatom Phaeodactylum tricornutum . By emphasizing biochemical approaches, novel insights in diatom biology have been realized. Better resolution of chrysolaminarin content from cell extracts provides not only an opportunity to study diatom carbon partitioning in more detail, but also the ability to specifically identify and characterize mutants with disrupted chrysolaminarin accumulation. Leveraging the latter point, I also studied a suite of gene knockouts and collected observations that indicate that Phatrdraft_50444 is the primary contributor to chrysolaminarin biosynthesis. Finally, I attempted to establish evidence for novel chrysolaminarin-related proteins by protein-carbohydrate affinity, which provided insights for future research strategies to explore these interactions. As such, the following chapters advance our understanding of chrysolaminarin metabolism and the general biology of the biotechnologically relevant diatom Phaeodactylum tricornutum . v ACKNOWLEDGMENTS First, I would like to thank my advisor, Graham Peers, for his ongoing mentorship over the last six years. Graham empowered me with a valuable opportunity to explore and learn from my mistakes along the way. He has helped me stay the course during my PhD studies. I will never forget his support during my particularly difficult Spring 2014 semester. I would also like to thank my committee, John Belisle, Dan Bush, and Ashok Prasad for their feedback during committee meetings, attending my CMB seminars, and clearly showing genuine interest in my academic development. I always appreciated bumping into you on campus. I am humbled to thank many individuals for their time and effort in various collaborations. Naoki Sato kindly welcomed me as a visiting summer graduate student at the University of Tokyo in 2012 and trained me how to analyze cyanobacteria lipid profiles using thin layer chromatography and GC. Mam Sherman in the Protein Expression and Purification Facility at CSU trained me on FPLC for protein purification, and helped me troubleshoot issues with recombinant protein expression. Libin Shi in the Delphi Chatterjee lab trained me on how to prepare and analyze GC/MS carbohydrate profiles. Chris Rithner in the Central Instrument Facility ran the 1H-NMR analysis of chrysolaminarin, which we interpreted together. Denis Jallet trained me on the Walz PAM fluorometer and programmed scripts to investigate the photophysiology of Phaeodactylum as well as quantify triacylglycerol via thin layer chromatography . Denis Jallet also facilitated the transfer of the CRISPR genome editing methods for Phaeodactylum from our collaborators at JCVI, Andrew Allen and Mark Moosburner. David Xing assembled the Phaeodactylum cDNA expression library, which I was fortunately able to vi apply to my project. Corey Brockling ran the targeted lipidomics experiment, and guided me through interpreting the data. I am grateful for this tremendous collective effort. I am grateful to the National Science Foundation which supported me financially during most of graduate school through the Graduate Research Fellowship and the Integrative Graduate
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