Optimal conditions of algal breeding using neutral beam and applying it to breed Euglena gracilis strains with improved lipid accumulation.

Microalgae are considered to be more useful and effective to use in biomass production than other photosynthesis organisms. However, microalgae need to be altered to acquire more desirable traits for the relevant purpose. Although neutron radiation is known to induce DNA mutations, there have been few studies on its application to microalgae, and the optimal relationship between irradiation intensity and mutation occurrence has not been established. In this study, using the unicellular red alga Cyanidioschyzon merolae as a model, we analyzed the relationship between the absorbed dose of two types of neutrons, high-energy (above 1 MeV) and thermal (around 25 meV) neutrons, and mutation occurrence while monitoring mutations in URA5.3 gene encoding UMP synthase. As a result, the highest mutational occurrence was observed when the cells were irradiated with 20 Gy of high-energy neutrons and 13 Gy of thermal neutrons. Using these optimal neutron irradiation conditions, we next attempted to improve the lipid accumulation of Euglena gracilis, which is a candidate strain for biofuel feedstock production. As a result, we obtained several strains with a maximum 1.3-fold increase in lipid accumulation compared with the wild-type. These results indicate that microalgae breeding by neutron irradiation is effective.

The biomolecules of Euglena gracilis: Harnessing biology for natural solutions to future problems.

Over the past decade, the autotrophic and heterotrophic protist Euglena gracilis (E. gracilis) has gained popularity across the studies of environmental science, biosynthesis experiments, and nutritional substitutes. The unique physiology and versatile metabolism of E. gracilis have been a recent topic of interest to many researchers who continue to understand the complexity and possibilities of using E. gracilis biomolecule production. In this review, we present a comprehensive representation of recent literature outlining the various uses of biomolecules derived from E. gracilis across the fields of natural product biosynthesis, as a nutritional substitute, and as bioremediation tools. In addition, we highlight effective strategies for altering metabolite production using abiotic stressors and growth conditions. To better understand metabolite biosynthesis and its role in E. gracilis, integrated studies involving genomics, metabolomics, and proteomics should be considered. Together, we show how the ongoing advancements in E. gracilis related research continue to broaden applications in the biosynthetic sector and highlight future works that would strengthen our understanding of overall Euglena metabolism.

The impact of elevated sulfur and nitrogen levels on cadmium tolerance in Euglena species.

Heavy metal (HM) pollution threatens human and ecosystem health. Current methods for remediating water contaminated with HMs are expensive and have limited effect. Therefore, bioremediation is being investigated as an environmentally and economically viable alternative. Freshwater protists Euglena gracilis and Euglena mutabilis were investigated for their tolerance to cadmium (Cd). A greater increase in cell numbers under Cd stress was noted for E. mutabilis but only E. gracilis showed an increase in Cd tolerance following pre-treatment with elevated concentrations of S or N. To gain insight regarding the nature of the increased tolerance RNA-sequencing was carried out on E. gracilis. This revealed transcript level changes among pretreated cells, and additional differences among cells exposed to CdCl2. Gene ontology (GO) enrichment analysis reflected changes in S and N metabolism, transmembrane transport, stress response, and physiological processes related to metal binding. Identifying these changes enhances our understanding of how these organisms adapt to HM polluted environments and allows us to target development of future pre-treatments to enhance the use of E. gracilis in bioremediation relating to heavy metals.

Metabolic responses of Euglena gracilis under photoheterotrophic and heterotrophic conditions.

The protist Euglena gracilis has various trophic modes including heterotrophy and photoheterotrophy. To investigate how cultivation mode influences metabolic regulation, the chemical composition of cellular metabolites of Euglena gracilis grown under heterotrophic and photoheterotrophic conditions was monitored from the early exponential phase to the mid-stationary phase using two different techniques, i.e, nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The combined metabolomics approach allowed an in-depth understanding of the mechanism of photoheterotrophic and heterotrophic growth for biomolecule production. Heterotrophic conditions promoted the production of polar amino and oxygenated compounds such as proteins and polyphenol compounds, especially at the end of the exponential phase while photoheterotrophic cells enhanced the production of organoheterocyclic compounds, carbohydrates, and alkaloids.

Transcriptome analysis of the effects of different carbon dioxide concentrations on paramylon accumulation in Euglena gracilis Z.

Appropriate concentration of carbon dioxide (CO2) will promote algae growth and metabolism. Building upon this finding, the present study investigated the impact of different CO2 concentrations (5% and 20%) on the carbon sequestration capacity of E. gracilis through aeration culturing, employing a combination of physiological analyses and transcriptome analysis. The results demonstrated that under 5% CO2 concentration, the cell density of E. gracilis was 1.79 times higher than that achieved in an air culture condition, and the paramylon content of E. gracilis was found to be 6.18 times higher than that of the air group. Based on transcriptome analysis, the carbon metabolism of E. gracilis was discussed. Significant up-regulation expression of genes associated with carbon synthesis was validated by an increase in paramylon content. This study revealed that under 5% CO2 conditions, E. gracilis exhibited elevated growth rate and enhanced photosynthetic carbon assimilation efficiency.

Understanding wax ester synthesis in Euglena gracilis: Insights into mitochondrial anaerobic respiration.

Euglena gracilis, photosynthetic protist, has a unique ability to generate wax esters in the absence of oxygen, employing a distinctive fatty acid synthesis mechanism. Through comprehensive inhibitor assays and gene-silencing techniques, our research clearly emphasized the indispensable role of the mitochondrial anaerobic respiratory chain in this biosynthesis. We identified acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) as central molecular components in the pathway. These findings strongly indicated a potential reversal of beta-oxidation occurring within mitochondria for fatty acid production in anaerobic conditions. Furthermore, our analysis revealed the pivotal function of nicotinamide nucleotide transhydrogenase (NNT) in efficiently managing the NADPH/NAD+ conversion essential for sustaining anaerobic metabolism. This review outlines our key findings and provides a comprehensive understanding of the molecular mechanisms that enable E. gracilis to produce wax ester anaerobically.

Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins.

Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.

Transcriptomic and genomic identification of spliceosomal genes from Euglena gracilis.

Diverse splicing types in nuclear and chloroplast genes of protist Euglena gracilis have been recognized for decades. However, the splicing machinery responsible for processing nuclear precursor messenger RNA introns, including trans-splicing of the 5' terminal outron and spliced leader (SL) RNA, remains elusive. Here, we identify 166 spliceosomal protein genes and two snRNA genes from E. gracilis by performing bioinformatics analysis from a combination of next-generation and full-length transcriptomic RNA sequencing (RNAseq) data as well as draft genomic data. With the spliceosomal proteins we identified in hand, the insensitivity of E. gracilis to some splicing modulators is revealed at the sequence level. The prevalence of SL RNA-mediated trans-splicing is estimated to be more than 70% from our full-length RNAseq data. Finally, the splicing proteomes between E. gracilis and its three evolutionary cousins within the same Excavata group are compared. In conclusion, our study characterizes the spliceosomal components in E. gracilis and provides the molecular basis for further exploration of underlying splicing mechanisms.

Euglena gracilis: Biochemical properties of a membrane bound ecto-phosphatase activity modulated by fluoroaluminate complexes and different trophic conditions.

The ecto-phosphatases belong to a group of enzymes closely associated with the cell surface that has its catalytic site facing the extracellular environment, where different phosphorylated substrates can be hydrolyzed. In the present work, we biochemically characterized the ecto-phosphatase activity of the freshwater microalgae Euglena gracilis, a model microorganism, ubiquitously distributed and resistant to several environmental stressors. The ecto-phosphatase activity is acidic, stimulated by copper and presents the following apparent kinetic parameters: Km = 2.52 ± 0.12 mM p-NPP and Vmax = 3.62 ± 0.06 nmol p-NP × h-1 × 106 cells. We observed that zinc, orthovanadate, molybdate, fluoride, and inorganic phosphate inhibit the ecto-phosphatase activity with different magnitudes. Fluoroaluminate complexes are also inhibitors of this ecto-phosphatase activity. They can be formed in the enzyme reaction conditions and are likely to occur in a natural environment where E. gracilis can be found. The ecto-phosphatase activity is constant through the culture growth phases and is negatively modulated after continuous subculturing in the dark when a shift from phototrophic to the heterotrophic metabolism is likely. The analysis of those biochemical parameters may contribute to understanding the role of E. gracilis ecto-phosphatase activity in natural environments.

Metabolomic analysis and pathway profiling of paramylon production in Euglena gracilis grown on different carbon sources.

Paramylon (ß-1,3-glucan) produced by Euglena gracilis displays antioxidant, antitumor, and hypolipidaemic functions. The biological properties of paramylon production by E. gracilis can be understood by elucidating the metabolic changes within the algae. In this study, the carbon sources in AF-6 medium were replaced with glucose, sodium acetate, glycerol, or ethanol, and the paramylon yield was measured. Adding 0.1260 g/L glucose to the culture medium resulted in the highest paramylon yield of 70.48 %. The changes in metabolic pathways in E. gracilis grown on glucose were assessed via non-targeted metabolomics analysis using ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-Orbitrap mass spectrometry. We found that glucose, as a carbon source, regulated some differentially expressed metabolites, including l-glutamic acid, γ-aminobutyric acid (GABA), and l-aspartic acid. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes further showed that glucose regulated the carbon and nitrogen balance through the GABA shunt, which enhanced photosynthesis, regulated the flux of carbon and nitrogen into the tricarboxylic acid cycle, promoted glucose uptake, and increased the accumulation of paramylon. This study provides new insights into E. gracilis metabolism during paramylon synthesis.

Fast and efficient molecule delivery into Euglena gracilis mediated by cell-penetrating peptide or dimethyl sulfoxide.

This study describes the development of two methods for delivering exogenous materials into Euglena gracilis, a unicellular flagellate organism. We report that the use of Pep-1, a short cell-penetrating peptide (CPP), or dimethyl sulfoxide (DMSO) can mediate fast and efficient intracellular delivery of exogenous materials into E. gracilis, achieving cellular entry efficiency as high as 70-80%. However, compared with human cells, the penetration of this algal cell with CPP requires a much higher concentration of purified proteins. In addition, upon convenient treatment with DMSO, E. gracilis cells can efficiently adsorb exogenous proteins and DNA with 10% DMSO as the optimal concentration for Euglena cells. Our results provide more options for the E. gracilis transformation 'toolkit box' and will facilitate future molecular manipulations of this microalgal organism.

Gravity sedimentation of eukaryotic algae Euglena gracilis accelerated by ethanol cultivation.

Euglena gracilis (E. gracilis) is a unicellular microalga with various applications in medicine, agriculture, aquaculture, health supplement, and jet fuel production. Euglena possibly solves population growth and exhaustion of fossil resources. Efficient cell harvesting is needed for the industry, and the gravity sedimentation method is low cost and does not require any equipment, although it has low efficiency. This study showed that the gravity sedimentation of E. gracilis cells is improved by cultivation in the presence of ethanol (EtOH). The gravity sedimentation of E. gracilis cells cultivated under 0.5% or 1.0% EtOH conditions was faster than that cultivated without EtOH. The mean calculated cell diameter was also found to be largest in cells cultivated under 0.5% or 1.0% EtOH conditions compared to that in cells cultivated without EtOH. Intracellular paramylon content, cell shapes, and motility differed between cells cultivated under 0.5% or 1.0% EtOH conditions and in the absence of EtOH. The results suggest that E. gracilis cultivation with EtOH leads to increased cell productivity, paramylon production, and efficient cell harvesting. KEY POINTS: • Euglena gracilis is an edible microalga producing value-added metabolites. • Ethanol addition upregulates E. gracilis growth and paramylon accumulation. • Gravity sedimentation is accelerated by ethanol-grown E. gracilis cells.

Versatile biotechnological applications of Euglena gracilis.

Euglena gracilis is a freshwater protist possessing secondary chloroplasts of green algal origin. Various physical factors (e.g. UV) and chemical compounds (e.g. antibiotics) cause the bleaching of E. gracilis cells-the loss of plastid genes leading to the permanent inability to photosynthesize. Bleaching can be prevented by antimutagens (i.e. lignin, vitamin C and selenium). Besides screening the mutagenic and antimutagenic activity of chemicals, E. gracilis is also a suitable model for studying the biological effects of many organic pollutants. Due to its capability of heavy metal sequestration, it can be used for bioremediation. E. gracilis has been successfully transformed, offering the possibility of genetic modifications for synthesizing compounds of biotechnological interest. The novel design of the "next generation" transgenic expression cassettes with respect to the specificities of euglenid gene expression is proposed. Moreover, E. gracilis is a natural source of commercially relevant bioproducts such as (pro)vitamins, wax esters, polyunsaturated fatty acids and paramylon (ß-1,3-glucan). One of the highest limitations of large-scale cultivation of E. gracilis is its disability to synthesize essential vitamins B1 and B12. This disadvantage can be overcome by co-cultivation of E. gracilis with other microorganisms, which can synthesize sufficient amounts of these vitamins. Such co-cultures can be used for the effective accumulation and harvesting of Euglena biomass by bioflocculation.

Cytochrome P450 Surface Domains Prevent the ß-Carotene Monohydroxylase CYP97H1 of Euglena gracilis from Acting as a Dihydroxylase.

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, ß-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated ß-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

Evaluation of the Effects of Euglena gracilis on Enhancing Immune Responses in RAW264.7 Cells and a Cyclophosphamide-Induced Mouse Model.

In this study we evaluated the immune-enhancing effects of ß-glucan, the main component of Euglena gracilis (Euglena), and Euglena on inflammatory factor expression in RAW264.7 macrophages and ICR mice with cyclophosphamide-induced immunosuppression. Macrophages were treated with ß-glucan or Euglena for 48 h. The ß-glucan and Euglena groups exhibited higher levels of inducible nitric oxide synthase, nitric oxide, and tumor necrosis factor (TNF)-α than the control (vehicle alone) group. Animals were fed saline and ß-glucan (400 mg/kg body weight (B.W.)) or Euglena (400 or 800 mg/kg B.W.) for 19 days, and on days 17-19, cyclophosphamide (CCP, 80 mg/kg B.W.) was administered to induce immunosuppression in the ICR mouse model. CCP reduced the body weight, spleen index, and cytokine expression of the mice. To measure cytokine and receptor expression, splenocytes were treated with concanavalin A (ConA) or lipopolysaccharide (LPS) as a mitogen for 24 h. In vivo, ConA stimulation significantly upregulated the expression of interferon (IFN)-γ, interleukin (IL)-10, IL-12 receptor ß1, IL-1ß, and IL-2 in splenocytes from the ß-glucan- or Euglena-treated groups compared with those in the splenocytes from the CCP-treated group; LPS stimulation increased the levels of the cytokines TNF-α, IL-1ß, and IL-6 in splenocytes from the ß-glucan- or Euglena-treated groups compared with those from the CCP-treated group, but most of these differences were not significant. These results demonstrate the effect of Euglena in ameliorating macrophages and immunosuppression in CCP-treated mice. Thus, Euglena has the potential to enhance macrophage- and splenocyte-mediated immune-stimulating responses.

Phytohormones enhance heavy metal responses in Euglena gracilis: Evidence from uptake of Ni, Pb and Cd and linkages to hormonomic and metabolomic dynamics.

Over the last decade, significant effort has been made to understand phytohormonal functions (e.g., cytokinins (CKs) and abscisic acid (ABA)) in metal stress responses of higher plants and algae. Despite the potential for these phytohormones to improve industrial remediation by Euglena gracilis (Euglenophyceae), no such roles have been elucidated for this highly adaptive species and its response to heavy metals. This study demonstrates that toxic metals (nickel, lead, cadmium) modify hormonal activity profiles (i.e., CK forms and their concentrations) in E. gracilis. Furthermore, exogenous ABA or CK (tZ) enabled higher metal uptake efficiency (i.e., 9.35% in lead and 9.2% in cadmium uptake with CK) and alleviated metal toxicity through the regulation of endogenous CKs (i.e., total CK, isoprenoid CK) and gibberellin (GAs, GA1 and GA3) levels. These responses suggest that E. gracilis regulates multiple phytohormone signals during metal stress acclimation. A deeper approach, using untargeted metabolomic analyses, gave more detailed insight into phytohormone-controlled pathways and associated modified metabolites, which were frequently related to metal accumulation and the physiological acclimation to metal presence. Significant changes in the levels of cellular metabolites, especially those involved in acclimation to metal stress, were under the influence of phytohormones in algal cells. When grown under metal stress conditions, the presence of exogenous ABA or CKs, caused changes in cellular metabolites which included those from: lipid pathways, riboflavin metabolism, the biosynthesis of cofactors/vitamins, and carbohydrate metabolism. Also, bioactive secondary metabolites (e.g., terpenoids, alkaloids, flavonoids, carotenoids) were modified in algal cells treated with phytohormones. Thus, the study gives a detailed view on the regulatory functions of ABA and CKs in algal metal bioremediation strategies, which are attributed to enhanced metal uptake and in the fine-tuning of plant hormone levels during metal stress response. The results can guide efforts to develop efficient, low-cost and environmentally friendly methods for bioremediation.

Glycosylated proteins in the protozoan alga Euglena gracilis: a proteomic approach.

Protein glycosylation, and in particular N-linked glycans, is a hallmark of eukaryotic cells and has been well-studied in mammalian cells and parasites. However, little research has been conducted to investigate the conservation and variation of protein glycosylation pathways in other eukaryotic organisms. Euglena gracilis is an industrially important microalga, used in the production of biofuels and nutritional supplements. It is evolutionarily highly divergent from green algae and more related to kinetoplastid pathogens. It was recently shown that E. gracilis possesses the machinery for producing a range of protein glycosylations and make simple N-glycans, but the modified proteins were not identified. This study identifies the glycosylated proteins, including transporters, extracellular proteases, and those involved in cell surface signalling. Notably, many of the most highly expressed and glycosylated proteins are not related to any known sequences and are, therefore, likely to be involved in important novel functions in Euglena.

Microbe Profile: Euglena gracilis: photogenic, flexible and hardy.

Euglena gracilis is a unicellular photosynthetic eukaryotic flagellate of the Discoba supergroup, which also encompasses Kinetoplastida and Diplonema. Plastids have green algal origin and are secondarily acquired. The nuclear genome is extremely large and many genes suggest multiple endosymbiotic/gene transfer events, i.e. derivation from prokaryotes of various lineages. E. gracilis is remarkably robust and can proliferate in environments contaminated with heavy metals and acids. Extraordinary metabolic plasticity and a mixotrophic lifestyle confers an ability to thrive in a broad range of environments, as well as facilitating production of many novel metabolites, making Euglena of considerable biotechnological importance.

Mixotrophic Cultivation Optimization of Microalga Euglena pisciformis AEW501 for Paramylon Production.

Euglena, a flagellated unicellular protist, has recently received widespread attention for various high-value metabolites, especially paramylon, which was only found in Euglenophyta. The limited species and low biomass of Euglena has impeded paramylon exploitation and utilization. This study established an optimal cultivation method of Euglena pisciformis AEW501 for paramylon production under mixotrophic cultivation. The results showed that the optimum mixotrophic conditions were 20 °C, pH 7.0, and 63 µmol photons m-2∙s-1, and the concentrations of sodium acetate and diammonium hydrogen phosphate were 0.98 g L-1 and 0.79 g L-1, respectively. The maximal biomass and paramylon content were 0.72 g L-1 and 71.39% of dry weight. The algal powder contained more than 16 amino acids, 6 vitamins, and 10 unsaturated fatty acids under the optimal cultivation. E. pisciformis paramylon was pure ß-1,3-glucan-type polysaccharide (the purity was up to 99.13 ± 0.61%) composed of linear glucose chains linked together by ß-1,3-glycosidic bonds. These findings present a valuable basis for the industrial exploitation of paramylon with E. pisciformis AEW501.

Metabolomics revealed the photosynthetic performance and metabolomic characteristics of Euglena gracilis under autotrophic and mixotrophic conditions.

Photosynthetic and metabolomic performance of Euglena gracilis was examined and compared under autotrophic and mixotrophic conditions. Autotrophic protozoa (AP) obtained greater biomass (about 33% higher) than the mixotrophic protozoa (MP) after 12 days of growth. AP maintained steady photosynthesis, while MP showed a remarkable decrease in photosynthetic efficiency and dropped to an extremely low level at day 12. In MP, low light absorption and photosynthetic electron transport efficiency, and high energy dissipation were reflected by the chlorophyll (chl a) fluorescence (OJIP) of the protozoa. The values of ΨO, ΦEo, and ETO/RC of MP decreased to extremely low levels, to 1/15, 1/46, and 1/9 those of AP, respectively, while DIO/RC increased to approximately 16 times that of AP. A total of 137 metabolites were showed significant differences between AP and MP. AP accumulated more monosaccharide, lipids, and alkaloids, while MP produced more amino acids, peptides, and long-chain fatty acids including poly-unsaturated fatty acids. The top nine most important enriched pathways obtained from KEGG mapping were related to ABC transporters, biosynthesis of amino acids, purine metabolism, and carbohydrate metabolism. There were significant differences between AP and MP in photosynthetic activity, metabolites, and metabolic pathways. This work presented useful information for the production of high value bioproducts in E. gracilis cultured under different nutritional conditions.