Application of label-free shotgun proteomics for the analysis of metabolic pathways in Euglena gracilis and its potential as a source of food supplements

Empirical thesis.Includes bibliographical references.Chapter 1. Introduction -- Chapter 2. Materials and methods -- Chapter 3. A comprehensive assessment of the biosynthetic pathways of ascorbate, α-tocopherol and free amino acids in Euglena gracilis var. saccharophila -- Chapter 4. Comparative proteomics investigation of central carbon metabolism in Euglena gracilis grown under photoautotrophic, mixotrophic and heterotrophic cultivations -- Chapter 5. Differential response of Euglena gracilis enzymes involved in paramylon metabolism under photoautotrophic, mixotrophic and heterotrophic cultivations -- Chapter 6. Investigation of Euglena gracilis var. saccharophila as a producer of valuable metabolites during mixotrophic cultivation in a laboratory-scale bioreactor -- Chapter 7. Conclusive summary and future research directions -- Chapter 11. Supporting information -- Appendix.Proteomic studies provide insight into the global expression patterns of proteins within cells. Proteomic profiling of the freshwater protist Euglena gracilis has not been previously investigated, although its transcriptome has recently been published. This work aimed at profiling and identifying proteins involved in carbon metabolism, the biosynthetic and degradation pathways of paramylon, and the biosynthetic pathways of α-tocopherol, ascorbate and the twenty protein-building amino acids under photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivations.Paramylon content was the highest under HT condition, with the streptomycin-bleached mutant E. gracilis ZSB synthesising the most paramylon and the wild-type E. gracilis var. saccharophila synthesising paramylon the fastest. However, antioxidant content was the highest under PT condition, with the wild-type E. gracilis Z strain accumulating the most antioxidants. The abundance of some free amino acids varied between the mid-exponential phase and the beginning of the stationary phase, and between growth conditions, but the total amount remained about the same with arginine as the most abundant amino acid.Label-free shotgun proteomics enabled identification of over 4000 translated proteins, about 30% of which could not be annotated by sequence similarity alone. Many enzymes exhibited several isoforms that were influenced by growth condition. Not all isozymes identified in the transcriptome were detected in the proteome, suggesting post-transcriptional regulation. The results indicated diversity of pathways similar to different organisms, such as lysine biosynthesis to fungi, and serine and proline biosyntheses to plants. Some pathways were unique to Euglena, such as the TCA cycle, and paramylon, ascorbate and arginine biosyntheses.The proteomic studies revealed that instead of hexokinase E. gracilis uses a high-specificity glucokinase for the EMP pathway. Two paramylon synthase candidates (EgGSL1 and EgGSL2) were identified of which EgGSL2 was predominant and expressed under all growth conditions. EgGSL1 was not expressed under HT condition, but the gene transcript was detected by qRT-PCR across all growth conditions indicating light induction and post-transcriptional regulation. Some enzymes of the Calvin pathway were expressed under HT cultivation suggesting post-translational regulation. HT cells may also carry out CO2 fixation in the dark even in the presence of sufficient glucose in the medium. Two pathways for serine biosynthesis were identified, one of which was prevalent under PT and MT cultivation, and the other under HT cultivation.The MT cultivation of E. gracilis var. saccharophila was further chosen for evaluation of this strain as a potential source of food supplements in a laboratory-scale bioreactor, as this strain produced higher paramylon than E. gracilis Z, and under MT cultivation its antioxidant levels were higher than those of E. gracilis ZSB.This work expands on the existing knowledge of metabolic pathways in E. gracilis and provides insight into how these pathways are influenced by growth condition, thus providing a foundation for future strain engineering.Mode of access: World wide web1 online resource (xvii, 190, ) diagrams, graphs, table

Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the ?-1,3-glucan paramylon under varying light conditions

Euglena gracilis Z and a “sugar loving” variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a β-1,3-glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose–yeast extract medium supporting high-level paramylon production. Both strains produced the highest paramylon yields (7.4–8 g · L−1, respectively) while grown in the dark, but the maximum yield was achieved faster by E. gracilis var. saccharophila (48 h vs. 72 h). The glucose-to-paramylon yield coefficient Ypar/glu = 0.46 ± 0.03 in the E. gracilis var. saccharophila cultivation, obtained in this study, is the highest reported to date. Proteomic analysis of the metabolic pathways provided molecular clues for the strain behavior observed during cultivation. For example, overexpression of enzymes in the gluconeogenesis/glycolysis pathways including fructokinase-1 and chloroplastic fructose-1,6-bisphosphatase (FBP) may have contributed to the faster rate of paramylon accumulation in E. gracilis var. saccharophila. Differentially expressed proteins in the early steps of chloroplastogenesis pathway including plastid uroporphyrinogen decarboxylases, photoreceptors, and a highly abundant (68-fold increase) plastid transketolase may have provided the E. gracilis var. saccharophila strain an advantage in paramylon production during diurnal cultivations. In conclusion, the variant strain E. gracilis var. saccharophila seems to be well suited for producing large amounts of paramylon. This work has also resulted in the identification of molecular targets for future improvement of paramylon production in E. gracilis, including the FBP and phosophofructokinase 1, the latter being a key regulator of glycolysis

A comprehensive assessment of the biosynthetic pathways of ascorbate, α-tocopherol and free amino acids in Euglena gracilis var. saccharophila

Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13 mg g⁻¹ ascorbate and 2.52 mg g⁻¹ α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50 mg g⁻¹ ascorbate, and 1.40 and 0.21 mg g⁻¹ α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 non-redundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals

Probing the Role of the Chloroplasts in Heavy Metal Tolerance and Accumulation in Euglena gracilis

The E. gracilis Zm-strain lacking chloroplasts, characterized in this study, was compared with the earlier assessed wild type Z-strain to explore the role of chloroplasts in heavy metal accumulation and tolerance. Comparison of the minimum inhibitory concentration (MIC) values indicated that both strains tolerated similar concentrations of mercury (Hg) and lead (Pb), but cadmium (Cd) tolerance of the Z-strain was twice that of the Zm-strain. The ability of the Zm-strain to accumulate Hg was higher compared to the Z-strain, indicating the existence of a Hg transportation and accumulation mechanism not depending on the presence of chloroplasts. Transmission electron microscopy (TEM) showed maximum accumulation of Hg in the cytosol of the Zm-strain and highest accumulation of Cd in the chloroplasts of the Z-strain indicating a difference in the ability of the two strains to deposit heavy metals in the cell. The highly abundant heavy metal transporter MTP2 in the Z-strain may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein highly increased in abundance in the Zm-strain could be a potential Hg transporter to either cytosol or mitochondria. Overall, the chloroplasts appear to have major role in the tolerance and accumulation of Cd in E. gracilis

Comparative proteomics investigation of central carbon metabolism in Euglena gracilis grown under predominantly phototrophic, mixotrophic and heterotrophic cultivations

Euglena gracilis can use a wide range of organic carbon sources, as well as CO2 from the atmosphere. This metabolic versatility is owed to the genome of E. gracilis that can encode a wide range of enzymes. Many of these enzymes are regulated post-transcriptionally, allowing the cells to adapt quickly to changes in their surroundings. Here we investigated the effect of predominantly phototrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on central carbon metabolism in E. gracilis Z using label-free shotgun proteomics. Differential expression between isozymes was observed based on the cultivation condition. A hexokinase enzyme identified in the published transcriptome was not detected in the proteome. Instead, a high-specificity glucokinase appeared to conduct the first step of glycolysis. Two candidates for paramylon synthase were identified (EgGSL1 and EgGSL2), of which the predominant EgGSL2 protein was detected across all growth conditions, while EgGSL1 was only detected in the presence of light (PT and MT cultivations). Proteomic analysis revealed that the oxidative pentose phosphate pathway also plays a key role in glucose metabolism under MT and HT cultivation. Some chloroplast-encoded proteins and enzymes of the Calvin pathway were detected under HT cultivation indicating regulation at the post-translational level. The carbon metabolic pathways investigated here in terms of proteomic changes provide new information, as well as validate data presented elsewhere with quantitative proteomics, adding to the existing knowledge of metabolism in E. gracilis. Putative functional annotations of several proteins that were previously unidentified are also provided

A Proteomic View of Cellular and Molecular Effects of Cannabis

Cannabis (Cannabis sativa), popularly known as marijuana, is the most commonly used psychoactive substance and is considered illicit in most countries worldwide. However, a growing body of research has provided evidence of the therapeutic properties of chemical components of cannabis known as cannabinoids against several diseases including Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease, schizophrenia and glaucoma; these have prompted changes in medicinal cannabis legislation. The relaxation of legal restrictions and increased socio-cultural acceptance has led to its increase in both medicinal and recreational usage. Several biochemically active components of cannabis have a range of effects on the biological system. There is an urgent need for more research to better understand the molecular and biochemical effects of cannabis at a cellular level, to understand fully its implications as a pharmaceutical drug. Proteomics technology is an efficient tool to rigorously elucidate the mechanistic effects of cannabis on the human body in a cell and tissue-specific manner, drawing conclusions associated with its toxicity as well as therapeutic benefits, safety and efficacy profiles. This review provides a comprehensive overview of both in vitro and in vivo proteomic studies involving the cellular and molecular effects of cannabis and cannabis-derived compounds