Changes in Gravitaxis and Gene-Expression in an Euglena gracilis Culture over Time.

Age-dependent changes in the transcription levels of 5-day-old Euglena gracilis cells, which showed positive gravitaxis, 6-day-old cells without gravitactic orientation, and older cells (9- and 11-day-old, which displayed a precise negative gravitaxis) were determined through microarray analysis. Hierarchical clustering of four independent cell cultures revealed pronounced similarities in transcription levels at the same culture age, which proves the reproducibility of the cultivation method. Employing the non-oriented cells from the 6-day-old culture as a reference, about 2779 transcripts were found to be differentially expressed. While positively gravitactic cells (5-day-old culture) showed only minor differences in gene expression compared to the 6-day reference, pronounced changes of mRNAs (mainly an increase) were found in older cells compared to the reference culture. Among others, genes coding for adenylyl cyclases, photosynthesis, and metabolic enzymes were identified to be differentially expressed. The investigated cells were grown in batch cultures, so variations in transcription levels most likely account for factors such as nutrient depletion in the medium and self-shading. Based on these findings, a particular transcript (e.g., transcript 19556) was downregulated using the RNA interference technique. Gravitaxis and phototaxis were impaired in the transformants, indicating the role of this transcript in signal transduction. Results of the experiment are discussed regarding the increasing importance of E. gracilis in biotechnology as a source of valuable products and the possible application of E. gracilis in life-support systems.

[Advances of studies on culture and product functions of Euglena gracilis].

Euglena gracilis is a unicellular eukaryote between animal and plant cells, which is widely distributed in nature. E. gracilis has both plant and animal characteristics, and can grow photoautotrophically, heterotrophically and mixotrophically. E. gracilis also features on abundant and various cellular composition. Recently, extensive researches on unique cellular components of E. gracilis have revealed its application in the field of medicine, food, and feedstuff, in terms of improving immunity, fighting inflammation, and lowering uric acid levels. The application prospects of paramylon in biomedical area were also discovered. As food ingredients, food additives, feedstuffs and cosmetic ingredients, E. gracilis has been certified domestically and overseas. A series of products have been developed overseas, especially in Japan. However, the research and development of E. gracilis are still in its infancy in China, and there is huge space for development. At present, the research and potential application of cultivation and product functions of E. gracilis have been rarely reviewed. This review systematically examines both the domestic and abroad research of cultivation and production of E. gracilis, as well as the biological activity of E. gracilis powder and paramylon. The existing problems in the application, exploitation, and possible development direction of E. gracilis in the future are prospected. This review might be useful for establishing and optimizing large-scale and efficient heterotrophic technology, as well as developing related products of E. gracilis with specific functions.

High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.

Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

Nuclear mutagenesis and adaptive evolution improved photoautotrophic growth of Euglena gracilis with flue-gas CO2 fixation.

To effectively improve biomass growth and flue-gas CO2 fixation of microalgae, acid-tolerant Euglena gracilis was modified with cobalt-60 γ-ray irradiation and polyethylene glycol (PEG) adaptive screening to obtain the mutant strain M800. The biomass dry weight and maximum CO2 fixation rate of M800 were both 1.47 times higher than that of wild strain, which was attributed to a substantial increase in key carbon fixation enzyme RuBisCO activity and photosynthetic pigment content. The high charge separation quantum efficiency in PSII reaction center, efficient light utilization and energy regulation that favors light conversion, were the underlying drivers of efficient photosynthetic carbon fixation in M800. M800 had stronger antioxidant capacity in sufficient high-carbon environment, alleviating lipid peroxidation damage. After adding 1 mM PEG, biomass dry weight of M800 reached 2.31 g/L, which was 79.1 % higher than that of wild strain. Cell proliferation of M800 was promoted, the apoptosis and necrosis rates decreased.

Ras superfamily GTPases and signal transduction in Euglena gracilis.

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology.

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.

Fed-batch cultivation of Euglena gracilis for the high-yield production and GPC-assisted molecular weight determination of paramylon.

Paramylon, a starch-like carbohydrate accumulated in Euglena gracilis cells, could be a potential source as a raw material for chemical products; its high-yield production would thus be highly desired. Although the molecular weight and polymerization degree of paramylon are important properties for its use as a raw material for chemical products, the available information about paramylon molecular weight remains insufficient. Therefore, in this study, we investigated a high-density E. gracilis culture approach and how culture conditions affect paramylon molecular weight. The nitrogen source, cultivation temperature, and nutrient feeding were optimized for maximum biomass and paramylon productivity. The maximum dry cell weight and paramylon content yields reached 108.9 g/L and 87.2%, respectively. Paramylon molecular weight was in the range of 220 000-320 000 Da. Our gel permeation chromatography analysis showed that the cells with a higher paramylon content tended to contain paramylon of a higher molecular weight.

Effects of polystyrene microplastics on Euglena gracilis: Intracellular distribution and the protozoan transcriptional responses.

Microplastics (MPs) introduced into aquatic environments inevitably interact with aquatic organisms such as plankton, potentially yielding adverse effects on the aquatic ecosystem. The extent to which MPs can infiltrate planktonic cells and evoke a molecular response remains largely unknown. In the present study, the internalization of fluorescently labeled polystyrene (PS) MPs on Euglena gracilis cells was investigated, determining the transcriptional responses within protozoa after an 8-day exposure period. The results showed that exposure to 25 mg/L PS-MPs for 8 days, significantly inhibited protozoan growth (P < 0.05) and decreased the chlorophyll a content of E. gracilis. The photosynthetic efficiency of E. gracilis was suppressed by MPs after 4 days, and then recovered to control values by the eighth day. Fluorescence imaging confirmed the presence of MPs in E. gracilis. Transcriptomic analysis revealed the influence of PS-MPs on a diverse range of transcriptional processes, encompassing oxidative phosphorylation, oxidation-reduction process, photosynthesis, and antioxidant enzymes. Notably, a majority of the differentially expressed genes (DEGs) exhibited down-regulation. Furthermore, PS-MPs disturbed the transcriptional regulation of chloroplasts and photosynthesis. These findings indicate a direct interaction between PS-MPs and organelles within E. gracilis cells following internalization, thereby disrupting regular gene expression patterns and posing a substantial environmental risk to the aquatic ecosystem.

Valorization of fruit and vegetable byproducts for the beta-glucan production from Euglena gracilis.

Five fruit and vegetable byproducts were evaluated as carbon sources and media for beta-glucan production from Euglena gracilis. Orange peel showed the highest beta-glucan concentration (6.5 g/L) and productivity (1.9 g/L/day) when used as a medium. However, when employed as carbon sources, apple pomace showed the highest beta-glucan concentration (10.6 g/L) and productivity (3.5 g/L/day). The appropriate chemical oxygen demand/nitrogen ratio (71.1) and favorable carbon sources of apple contributed to beta-glucan production. Increasing sugar concentrations in apple pomace and orange peel from 10 to 30 g/L raised the beta-glucan concentration to 11.6 g/L. Using apple pomace and orange peel individually proved more effective than mixing them for beta-glucan production. Therefore, apple as a carbon source is the most effective fruit and vegetables byproduct for beta-glucan production. This is expected to reduce the cost of E. gracilis cultivation on a large-scale and contribute to the circular economy.

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 (Euglena) powder supplementation enhanced immune function through natural killer cell activity in apparently healthy participants: A randomized, double-blind, placebo-controlled trial.

Euglena gracilis (Euglena) is a microalgae found in most freshwater environments that produces paramylon, an insoluble ß-1,3-glucan linked to human immunity. We hypothesized that Euglena powder has effects on immune function in apparently healthy adults. The study included male or female volunteers between the ages of 20 and 70 years who had white blood cell counts ranging from 4 × 103/µL to 10 × 103/µL, a "severe" rating on the stress questionnaire from the Korea National Health and Nutrition Examination Survey, and at least 2 upper respiratory infections with cold-like symptoms in the previous year. Participants received either a placebo or 700 mg of Euglena powder daily for 8 weeks. The study measured natural killer cell activity, cytokine concentrations, and blood lipid profiles to confirm the immune effect of Euglena consumption. In conclusion, Euglena improved immunological function through natural killer cell activity. Safety assessment showed no significant changes in vital signs or clinical chemistry indicators, and there were no adverse events associated with Euglena consumption. Euglena supplementation may help boost the immune systems of healthy individuals.

Euglena gracilis Extract Protects From Tobacco Smoke Carcinogen-Induced Lung Cancer by Altering Gut Microbiota Metabolome.

Extracts from Euglena gracilis have been shown to prevent cancer growth in mouse models. However, the molecular mechanism of this anti-cancer activity has not been determined nor has the effect of Euglena extracts on tobacco smoke carcinogen-induced carcinogenesis. Here, we investigate the hypothesis that this anti-cancer activity is a result of changes in the intestinal microbiota induced by oral administration of the extract. We found that a Euglena gracilis water extract prevents lung tumorigenesis induced by a tobacco smoke-specific carcinogen (NNK) in mice treated either 2 weeks before or 10 weeks after NNK injection. Both of these treatment regimens are associated with significant increases in 27 microbiota metabolites found in the mouse feces, including large increases in triethanolamine, salicylate, desaminotyrosine, N-acetylserine, glycolate, and aspartate. Increases in the short-chain fatty acids (SCFAs) including acetate, propionate and butyrate are also observed. We also detected a significant attenuation of lung carcinoma cell growth through the induction of cell cycle arrest and apoptosis caused by low levels of SCFAs. This study provides strong evidence of anti-cancer activity in Euglena gracilis extracts against tobacco smoke carcinogen-induced tumorigenesis and demonstrates that this activity is linked to increased production of specific gut microbiota metabolites and the resultant induction of cell cycle arrest and apoptosis of lung carcinoma cells.

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.

New insights into raceway cultivation of Euglena gracilis under long-term semi-continuous nitrogen starvation.

This study aimed to investigate the physiological responses of Euglena gracilis (E. gracilis) when subjected to semicontinuous N-starvation (N-) for an extended period in open ponds. The results indicated that the growth rates of E. gracilis under the N- condition (11 ± 3.3 g m-2 d-1) were higher by 23% compared to the N-sufficient (N+, 8.9 ± 2.8 g m-2 d-1) condition. Furthermore, the paramylon content of E.gracilis was above 40% (w/w) of dry biomass in N- condition compared to N+ (7%) condition. Interestingly, E. gracilis exhibited similar cell numbers regardless of nitrogen concentrations after a certain time point. Additionally, it demonstrated relatively smaller cell size over time, and unaffected photosynthetic apparatus under N- condition. These findings suggest that there is a tradeoff between cell growth and photosynthesis in E. gracilis, as it adapts to semi-continuous N- conditions without a decrease in its growth rate and paramylon productivity. Notably, to the author's knowledge, this is the only study reporting high biomass and product accumulation by a wild-type E. gracilis strain under N- conditions. This newly identified long-term adaptation ability of E. gracilis may offer a promising direction for the algal industry to achieve high productivity without relying on genetically modified organisms.

Glucans, Paramylon and Other Algae Bioactive Molecules.

Algae (macro- and micro-algae) can be defined as light-driven cell factories that synthesize bioactive compounds consisting of primary metabolites (i [...].

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.

Structural characterization of Euglena gracilis polysaccharide and its in vitro hypoglycemic effects by alleviating insulin resistance.

Diabetes mellitus, characterized by hyperglycemia and insulin resistance, is a disorder of the endocrine metabolic system which has emerged as a common chronic disease worldwide. Euglena gracilis polysaccharides have ideal development potential in the treatment of diabetes. However, their structure and bioactivity are largely unclear. A novel purified water-soluble polysaccharide (EGP-2A-2A) from E. gracilis with a molecular weight of 130.8 kDa consisted of xylose, rhamnose, galactose, fucose, glucose, arabinose, and glucosamine hydrochloride. The SEM image for EGP-2A-2A suggested a rough surface with the presence of globule-like protrusions. Methylation and NMR spectral analyses revealed that EGP-2A-2A was mainly composed of →6)-ß-D-Galp-(1 â†’ 2)-α-D-Glcp-(1 â†’ 2)-α-L-Rhap-(1 â†’ 3)-α-L-Araf-(1 â†’ 6)-ß-D-Galp-(1 â†’ 3)-α-D-Araf-(1 â†’ 3)-α-L-Rhap-(1 â†’ 4)-ß-D-Xylp-(1 â†’ 6)-ß-D-Galp-(1 â†’ with complex branching structure. EGP-2A-2A significantly increased glucose consumption and glycogen content in IR-HeoG2 cells and modulates glucose metabolism disorders by regulating PI3K, AKT, and GLUT4 signaling pathways. EGP-2A-2A significantly suppressed TC, TG, and LDL-c levels, and enhanced that of HDL-c. EGP-2A-2A ameliorated abnormalities caused by disorders of glucose metabolism and the hypoglycemic activity of EGP-2A-2A may be mainly positively related to its high glucose content and the ß-configuration in the main chain. These results suggested that EGP-2A-2A played an important role in alleviating disorders of glucose metabolism through insulin resistance and has the potential for development as a novel functional food with nutritional and health benefits.