Highly efficient transgene-free targeted mutagenesis and single-stranded oligodeoxynucleotide-mediated precise knock-in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins.
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ABSTRACT:
SUBMITTER: Nomura T
PROVIDER: S-EPMC6790356 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
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Highly efficient transgene-free targeted mutagenesis and single-stranded oligodeoxynucleotide-mediated precise knock-in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins.
Nomura Toshihisa T Inoue Komaki K Uehara-Yamaguchi Yukiko Y Yamada Koji K Iwata Osamu O Suzuki Kengo K Mochida Keiichi K
Plant biotechnology journal 20190611 11
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Project description:Euglena gracilis (E. gracilis) has been proposed as one of the most attractive microalgae species for biodiesel and biomass production, which exhibits a number of shapes, such as spherical, spindle-shaped, and elongated. Shape is an important biomarker for E. gracilis, serving as an indicator of biological clock status, photosynthetic and respiratory capacity, cell-cycle phase, and environmental condition. The ability to prepare E. gracilis of uniform shape at high purities has significant implications for various applications in biological research and industrial processes. Here, we adopt a label-free, high-throughput, and continuous technique utilizing inertial microfluidics to separate E. gracilis by a key shape parameter-cell aspect ratio (AR). The microfluidic device consists of a straight rectangular microchannel, a gradually expanding region, and five outlets with fluidic resistors, allowing for inertial focusing and ordering, enhancement of the differences in cell lateral positions, and accurate separation, respectively. By making use of the shape-activated differences in lateral inertial focusing dynamic equilibrium positions, E. gracilis with different ARs ranging from 1 to 7 are directed to different outlets.
| S-EPMC5589772 | biostudies-literature
Project description:BackgroundMicroalgae can contribute to more than 40% of global primary biomass production and are suitable candidates for various biotechnology applications such as food, feed products, drugs, fuels, and wastewater treatment. However, the primary limitation for large-scale algae production is the fact that algae requires large amounts of fresh water for cultivation. To address this issue, scientists around the world are working on ways to reuse the water to grow microalgae so that it can be grown in successive cycles without the need for fresh water.ResultsIn this study, we present the results when we cultivate microalgae with cultivation water that is purified and reused. Specifically, we purify the cultivation water using an ultrafiltration membrane (UFM) treatment and investigate how this treatment affects: the biomass and biochemical components of the microalgae; characteristics of microalgae growth inhibitors; the mechanism whereby potential growth inhibitors are secreted (followed using metabolomics analysis); the effect of activated carbon (AC) treatment and advanced oxidation processes (AOPs) on the removal of growth inhibitors of Euglena gracilis. Firstly, the results show that E. gracilis can be only cultivated through two growth cycles with water that has been filtered and reused, and the growth of E. gracilis is significantly inhibited when the water is used a third time. Secondly, as the number of reused water cycles increases, the Cl- concentration gradually increases in the cultivation water. When the Cl- concentration accumulates to a level of fivefold higher than that of the control, growth of E. gracilis is inhibited as the osmolality tolerance range is exceeded. Interestingly, the osmolality of the reused water can be reduced by replacing NH4Cl with urea as the source of nitrogen in the cultivation water. Thirdly, E. gracilis secretes humic acid (HA)-which is produced by the metabolic pathways for valine, leucine, and isoleucine biosynthesis and by linoleic acid metabolism-into the cultivation water. Because HA contains large fluorescent functional groups, specifically extended π(pi)-systems containing C=C and C=O groups and aromatic rings, we were able to observe a positive correlation between HA concentration and the rate of inhibition of E. gracilis growth using fluorescence spectroscopy. Moreover, photosynthetic efficiency is adversely interfered by HA, thereby reductions in the synthetic efficiency of paramylon and lipid in E. gracilis. In this way, we are able to confirm that HA is the main growth inhibitor of E. gracilis. Finally, we verify that all the HA is removed or converted into nutrients efficiently by AC or UV/H2O2/O3 treatments, respectively. As a result of these treatments, growth of E. gracilis is restored (AC treatment) and the amount of biomass is promoted (UV/H2O2/O3 treatment).ConclusionsThese studies have important practical and theoretical significance for the cyclic cultivation of E. gracilis and for saving water resources. Our work may also provide a useful reference for other microalgae cultivation.
| S-EPMC8180174 | biostudies-literature
Project description:Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A-gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.
| S-EPMC8230907 | biostudies-literature
Project description:By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3 μM in Aspergillus fumigatus and 0.29 μM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products.
| S-EPMC9321709 | biostudies-literature
Project description:Euglena gracilis, a green microalga known as a potential candidate for jet fuel producers and new functional food resources, is highly tolerant to antibiotics, heavy metals, and other environmental stresses. Its cells contain many high-value products, including vitamins, amino acids, pigments, unsaturated fatty acids, and carbohydrate paramylon as metabolites, which change contents in response to various extracellular environments. However, mechanism insights into the cellular metabolic response of Euglena to different toxic chemicals and adverse environmental stresses were very limited. We extensively investigated the changes of cell biomass, pigments, lipids, and paramylon of E. gracilis under several environmental stresses, such as heavy metal CdCl2, antibiotics paromomycin, and nutrient deprivation. In addition, global metabolomics by Ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was applied to study other metabolites and potential regulatory mechanisms behind the differential accumulation of major high-valued metabolites. This study collects a comprehensive update on the biology of E. gracilis for various metabolic responses to stress conditions, and it will be of great value for Euglena cultivation and high-value [154mm][10mm]Q7metabolite production.
| S-EPMC8329484 | biostudies-literature
Project description:Euglena gracilis is a well-known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular belonging to the same eukaryotic phylum as the parasitic trypanosomatids. As its nuclear genome has proven difficult to sequence, reliable transcriptomes are important for functional studies. In this work, we assembled a new consensus transcriptome by combining sequencing reads from five independent studies. Based on a detailed comparison with two previously released transcriptomes, our consensus transcriptome appears to be the most complete so far. Remapping the reads on it allowed us to compare the expression of the transcripts across multiple culture conditions at once and to infer a functionally annotated network of co-expressed genes. Although the emergence of meaningful gene clusters indicates that some biological signal lies in gene expression levels, our analyses confirm that gene regulation in euglenozoans is not primarily controlled at the transcriptional level. Regarding the origin of E. gracilis, we observe a heavily mixed gene ancestry, as previously reported, and rule out sequence contamination as a possible explanation for these observations. Instead, they indicate that this complex alga has evolved through a convoluted process involving much more than two partners.
| S-EPMC8227486 | biostudies-literature
Project description:Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.
| S-EPMC5745450 | biostudies-literature
Project description:Dietary supplementation with nutrients able to control intestinal and systemic inflammation is of marketable interest. Indeed, gastrointestinal homeostasis plays a significant role in maintaining human health. In this setting, E. gracilis may sustain or promote human health, but the effects on the intestinal inflammatory milieu are not clear. In this study, we investigated the anti-inflammatory activity of E. gracilis and inferred possible mechanisms. Paramylon, crude, and fractionated extracts were obtained from E. gracilis grown in vitro. Phytoconstituents of the extracts were characterized using TLC and HPLC UV-Vis. The anti-inflammatory and antioxidant activities were investigated in primary human macrophages and an intestinal epithelial cell line (HT-29). The analysis of the extracts led to identifying β-carotene, neoxanthin, diadinoxanthin, canthaxanthin, and breakdown products such as pheophytins and pheophorbides. E. gracilis fractionated extracts reduced the production of tumor necrosis factor-α triggered by bacterial lipopolysaccharide (LPS) in the short and long terms. Pheophytin a and b and canthaxanthin increased the intracellular reducing potential and dampened the production of LPS-induced reactive oxygen species and lipid peroxidation, intracellular events usually involved in the perpetuation of chronic inflammatory disorders. This study rationalizes the role of specific extract fractions of E. gracilis in controlling LPS-driven intestinal inflammation.
| S-EPMC8537577 | biostudies-literature
Project description:Euglena gracilis Transcriptome or Gene expression
| PRJNA1049904 | ENA