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- All Subjects: Biochemistry
- Genre: Academic theses
Description
Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS.
ContributorsPaul, Robin (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins.
ContributorsYao, Cheng I Daniel (Author) / Vermaas, Wim (Thesis advisor) / Fromme, Petra (Committee member) / Roberson, Robert (Committee member) / Webber, Andrew (Committee member) / Arizona State University (Publisher)
Created2011
Description
Microscopic algae have been investigated extensively by researchers for decades for their ability to bioremediate wastewater and flue gas while producing valuable biomass for use as feed, fuel, fertilizer, nutraceutical, and other specialty products. Reports of the exciting commercial potential of this diverse group of organisms started appearing in the literature as early as the 1940’s. However, nearly 80 years later, relatively few successful commercial microalgae installations exist and algae have not yet reached agricultural commodity status. This dissertation examines three major bottlenecks to commercial microalgae production including lack of an efficient and economical cultivation strategy, poor management of volatile waste nutrients, and costly harvesting and post processing strategies. A chapter is devoted to each of these three areas to gain a better understanding of each bottleneck as well as strategies for overcoming them.
The first chapter demonstrates the capability of two strains of Scenedesmus acutus to grow in ultra-high-density (>10 g L-1 dry weight biomass) cultures in flat panel photobioreactors for year-round production in the desert Southwest with record volumetric biomass productivity. The advantages and efficiency of high-density cultivation are discussed. The second chapter focuses on uptake and utilization of the volatile components of wastewater: ammonia and carbon dioxide. Scenedesmus acutus was cultured on wastewater from both municipal and agricultural origin and was shown to perform significantly better on flue gas as compared to commercial grade CO2 and just as well on waste nutrients as the commonly used BG-11 laboratory culture media, all while producing up to 50% lipids of the dry weight biomass suitable for use in biodiesel. The third chapter evaluates the feasibility of using gravity sedimentation for the harvesting of the difficult-to-separate Scenedesmus acutus green algae biomass followed by microfluidization to disrupt the cells. Lipid-extracted biomass was then studied as a fertilizer for plants and shown to have similar performance to a commercially available 4-6-6 fertilizer. Based on the work from these three chapters, a summary of modifications are suggested to help current and future microalgae companies be more competitive in the marketplace with traditional agricultural commodities.
The first chapter demonstrates the capability of two strains of Scenedesmus acutus to grow in ultra-high-density (>10 g L-1 dry weight biomass) cultures in flat panel photobioreactors for year-round production in the desert Southwest with record volumetric biomass productivity. The advantages and efficiency of high-density cultivation are discussed. The second chapter focuses on uptake and utilization of the volatile components of wastewater: ammonia and carbon dioxide. Scenedesmus acutus was cultured on wastewater from both municipal and agricultural origin and was shown to perform significantly better on flue gas as compared to commercial grade CO2 and just as well on waste nutrients as the commonly used BG-11 laboratory culture media, all while producing up to 50% lipids of the dry weight biomass suitable for use in biodiesel. The third chapter evaluates the feasibility of using gravity sedimentation for the harvesting of the difficult-to-separate Scenedesmus acutus green algae biomass followed by microfluidization to disrupt the cells. Lipid-extracted biomass was then studied as a fertilizer for plants and shown to have similar performance to a commercially available 4-6-6 fertilizer. Based on the work from these three chapters, a summary of modifications are suggested to help current and future microalgae companies be more competitive in the marketplace with traditional agricultural commodities.
ContributorsWray, Joshua (Author) / Dempster, Thomas (Thesis advisor) / Roberson, Robert (Thesis advisor) / Bingham, Scott (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2019
Description
The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this study show that indeed CEF is enhanced by the high amount of PSI present in WT. On the other hand, mutants with less PSI and less cyclic electron flow appeared able to maintain healthy levels of ATP synthesis through other compensatory mechanisms. Reduction in PSI abundance is naturally associated with reduced chlorophyll content, and mutants with less PSI showed greater primary productivity as light intensity increased due to increased light penetration in the cultures. Another question addressed in this research project involved the effect of deletion of flavoprotein 3 (an electron sink for PSI-generated electrons) from mutant strains that produce and secrete a fatty acid (Chapter 3). Removing Flv3 increased fatty acid production, most likely due to increased abundance of reducing equivalents that are key to fatty acid biosynthesis. Additional components of my dissertation research included examination of alkane biosynthesis in Synechocystis (Chapter 4), and effects of attempting to overexpress fibrillin genes for enhancement of stored compounds (Chapter 5). Synechocystis is an excellent platform for metabolic engineering studies with its photosynthetic capability and ease of genetic alteration, and the presented research sheds light on multiple aspects of its fundamental biology.
ContributorsMoore, Vickie (Author) / Vermaas, Willem (Thesis advisor) / Wang, Xuan (Committee member) / Roberson, Robert (Committee member) / Gaxiola, Roberto (Committee member) / Bingham, Scott (Committee member) / Arizona State University (Publisher)
Created2017
Description
Phenotypic and molecular profiling demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and the mutation frequency in basal-like subtype is as high as 80% and co-exists with several other somatic mutations in different genes. It was hypothesized that tumor heterogeneity is a result of a combination of neo-morphic functions of specific TP53 driver mutations and distinct co-mutations or the co-drivers for each type of TP53 mutation. The 10 most common p53 missense mutant proteins found in breast cancer patients were ectopically expressed in normal-like mammary epithelial cells and phenotypes associated with various hallmarks of cancer examined. Supporting the hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and polarity was observed in the mutants compared to wildtype p53 expressing cells. The missense mutants R248W, R273C and Y220C were most aggressive. Integrated analysis of ChIP and RNA seq showed distinct promoter binding profiles of the p53 mutant proteins different than wildtype p53, implying altered transcriptional activity of mutant p53 proteins and the phenotypic heterogeneity of tumors. Enrichment and model-based pathway analyses revealed dysregulated adherens junction and focal adhesion pathways associated with the aggressive p53 mutants. As several somatic mutations co-appear with mutant TP53, we performed a functional assay to fish out the relevant collaborating driver mutations, the co-drivers. When PTEN was deleted by CRISPR-Cas9 in non-invasive p53-Y234C mutant cell, an increase in cell invasion was observed justifying the concept of co-drivers. A genome wide CRISPR library-based screen on p53-Y234C and R273C cells identified separate candidate co-driver mutations that promoted cell invasion. The top candidates included several mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, the combined approach of molecular profiling and functional genomics screen highlighted distinct sets of co-driver mutations that can lead to heterogeneous phenotypes and promote aggressiveness in cells with different TP53 mutation background, which can guide development of novel targeted therapies.
ContributorsPal, Anasuya (Author) / LaBaer, Joshua (Thesis advisor) / Roberson, Robert (Committee member) / Van Horn, Wade (Committee member) / Maley, Carlo (Committee member) / Arizona State University (Publisher)
Created2019