De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions
© Yoshida et al. 2016
Received: 10 July 2015
Accepted: 25 February 2016
Published: 3 March 2016
The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions.
The E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel β-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment.
The RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.
KeywordsEuglena gracilis Wax ester fermentation Transcriptome analysis
Microalgae have recently attracted interest as a renewable source of biofuel and valuable compounds such as carotenoids, long chain unsaturated fatty acids, pigments, and polysaccharides [1, 2]. Microalgae are considered to have advantages over plants currently utilized for energy feedstock due to their fast growth rates, high lipid productivity, and cultivation on non-arable land areas that not compete with food production .
Euglena gracilis is a unicellular phytoflagellate that is widely distributed in freshwater, and has been proposed as a feedstock to produce biodiesel and various valuable compounds. For example, E. gracilis has been shown to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under optimal culture conditions E. gracilis has the ability to accumulate up to 50 % of paramylon per dry weight of the cells . Paramylon has potential applications not only in biomedical field due to its immunomodulation and anti-tumor activities, but also in industrial materials such as nanofibers [5, 6].
Paramylon is an important starting material for wax ester production under anaerobic conditions [7, 8]. When aerobically grown E. gracilis cells are transferred into anaerobic conditions, they degrade paramylon to actively synthesize and accumulate wax esters, which consist of medium-chain fatty acids and alcohols including 14:0 carbon chains as the major constituent. This phenomenon is designated wax ester fermentation due to the concomitant generation of ATP without any energy loss during wax ester production . Due to its low freezing point with a good Cetane number (66.2) , myristic acid (C14:0) has greater potential as a drop-in jet fuel than other algae produced medium-length fatty acids such as palmitic acid (C16:0) and stearic acid (C18:0).
The wax ester fermentation pathway proceeds across cellular compartments; glycolysis occurs in the cytoplasm, fatty acid biosynthesis in the mitochondria, and wax ester synthesis in the microsoms. The metabolic enzymes related to this pathway have biochemically analyzed. Anaerobic de novo fatty acid synthesis in mitochondria has been shown to utilize acetyl-CoA as a primer and a C2 donor, stemming from pyruvate via oxygen-labile pyruvate:NADP+ oxidoreductase instead of an ordinary pyruvate dehydrogenase complex . The pathway then proceeds via reversible enzymatic steps of β-oxidation by participation of a medium-chain tran-2-enoyl CoA reductase instead of acyl CoA dehydrogenase . However, the molecular details of most metabolic enzymes and the regulatory mechanism of wax ester fermentation in response to aerobic or anaerobic conditions remain largely unknown.
In addition to its advantage as a biofuel feedstock, E. gracilis is rich in nutrients such as vitamins, minerals, and well-balanced amino acids [12, 13]; therefore, it is used as a source of dietary supplement, in manufacture of cosmetics, and in the fortification of livestock feed. After the production of biodiesel by E. gracilis, the residual biomass can be converted into valuable industrial materials. Thus, E. gracilis is an ideal microalgal species for biodiesel and biomass production.
In spite of increasing interest in Euglena, genome and transcriptome data are not currently available, except for the chloroplast genome and a limited number of EST analyses [14–16]. An RNA-Seq analysis is one of the more superior molecular techniques being extensively used for the comprehensive gene expression profiling of living organisms including non-model organisms for which genomic information is currently limited, such as E.gracilis. In the present study, we performed RNA-Seq analysis to annotate functional transcripts and provide a comprehensive sequence resource for E.gracilis. Moreover, a comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions provided some insights into the regulation of wax ester metabolism.
Results and discussion
RNA-Seq and de novo assembly
Summary of E.gracilis RNA-Seq data
Read data (bps)
Total transcripts (seq)
FPKM≧1 out of total transcripts (seq)
FPKM≧1 out of components (seq)
Contig N50 (nucl)
Max length (nucl)
GC contents (%)
Components with SL-sequencea
14,186/26,479 (53.6 %)
Annotation of Euglena transcripts
Paramylon and wax ester metabolic pathway
Paramylon and wax esters are storage compounds in E.gracilis under aerobic and anaerobic conditions, respectively. Paramylon is synthesized from UDP-glucose via paramylon synthase (β-1,3-glucan synthase or callose synthase) . Based on its similarity to other β-1,3-glucan synthases, two components (comp36539_c1_seq4 and comp36157_c0_seq1) may be involved in the synthesis of paramylon (Additional file 1: Table S1). By comparing both FPKM values, the comp36157_c0_seq1 protein appears to be responsible for the synthesis due to its higher expression level than comp36539_c1_seq4. Previous studies reported that glucanase and phosphorylase were both involved in the degradation of paramylon [23, 24]. Seven components were annotated as β-1,3-glucanase; three of these components were recently purified from Euglena extract and identified as endo-form glucanase . On the other hand, no components with significant similarity to 1,3-beta-D-glucan phosphorylase were found in our assembled database.
Comparison of differentially expressed genes between E.gracilis cells in response to anaerobic conditions
Enriched GO terms of down-regulated genes in anaerobic state
macromolecule biosynthetic process
generation of precursor metabolites and energy
obsolete electron transport
Regarding expression changes in genes related to paramylon and wax ester metabolism, as described above, absolute fold-changes were less than 1.7-fold after 24 h anaerobic treatment (Additional file 1: Table S1). Furthermore, there was no consistency in most gene expression levels. For example, the expression levels for pyruvate:NADP+ oxidoreductase (PNO), which is considered to be a key regulatory enzyme for wax ester production due to its oxygen sensitivity , and for tran-2-enoyl CoA reductase 1 (TER1), were down-regulated even though we expected them to be up-regulated. Thus, when viewed globally, the gene expression changes that occurred during anaerobic treatment were not extensive or dynamic (Additional file 4). These limited expression changes do not appear to play a critical role in the activation of paramylon degradation and wax ester production under anaerobic conditions. In contrast to E.gracilis, a previous study reported that, in the diatom Phaeodactylum tricornutum, the transcriptional levels of some genes associated with glucan and fatty acid metabolism were altered in response to light/dark cycles, indicating significance of transcriptional regulation of central metabolic pathways . On the other hand, post-translational modifications, such as phosphorylation and acetylation, are known to play key roles in switching between respiratory and fermentative metabolism in response to aerobic and anaerobic conditions in yeast cells . Proteomic characterizations of post-translational modifications such as phosphorylation may provide important insights into the regulatory mechanism of wax ester fermentation in E.gracilis responding to changing oxygen conditions.
E.gracilis has atypical metabolic processes that provide extensive capacities for adaptation to extreme environmental conditions. One such process is a unique wax ester fermentation pathway activated under anaerobic conditions . In the present study, a de novo transcriptome analysis of E.gracilis was performed for the first time in an attempt to obtain a mechanistic understanding of anaerobic wax ester fermentation. The assembled and annotated sequence data identified the existing genes available from reference sequences and will benefit the Euglena research community. A comparative expression analysis showed that DEGs associated with energy metabolism, such as TCA cycle and oxidative phosphorylation, were altered in response to anaerobic conditions. Furthermore, this study obtained sequence information regarding enzymes potentially responsible for paramylon and wax ester metabolism. These sequences enabled us not only to elucidate the regulation mechanism of wax ester fermentation in response to changes in aeration conditions, but also to facilitate Euglena research by addressing basic biochemical, physiological, and evolutionary studies.
Euglena strain and culture
The wild-type E.gracilis strain Z was grown photoheterotrophically in Koren-Hutner (KH) medium  on a rotary shaker (120 rpm) under continuous light (100 μmol photons m−2 s−1) at 26 °C. Cell numbers were measured using the CASY Cell Counter and Analyzer System (Roche Applied Science, Basel, Switzerland). Stationary phase cells were incubated under anaerobic conditions in a sealed and shaded environment after N2-gas sparging for 5 min. The anaerobic state of cultures during experiments was confirmed by measuring oxygen concentration using an oxygen sensor (Fibox3, Taitec, Japan).
Total RNA was extracted from 1 mL of cultures (>106 cells) using RNAiso reagent (Takara, Japan), and purified using RNeasy Plus Universal Kits (Qiagen) following the manufacturer’s instructions. RNA quality and quantity were evaluated using Bioanalyzer 2100 (Agilent, CA).
RNA-Seq library preparation and sequencing
In order to obtain the Euglena reference sequence, an RNA-Seq library was constructed following the procedure recommended by Illumina. Briefly, mRNA was purified from 4 μg of the total RNA sample extracted from E.gracilis strain Z at stationary phase. It was then fragmented and converted to double-stranded DNA using TruSeq RNA Sample Preparation Kit v2 (Illumina). After a quality assessment and quantification of the sample libraries by Bioanalyzer 2100 (Agilent, CA) and a KAPA library quantification kit (KAPA Biosystems, UK), the library was sequenced using an Illumina HiSeq 2000 at the Exeter Sequencing Service at Exeter University. Next, sequencing for a detailed comparative gene expression analysis was performed using Illumina MiSeq after preparation of the RNA-Seq library from aerobic- and anaerobic-treated cells, following the same procedures as described above.
De novo transcriptome assembly
To obtain high-quality clean reads, the raw reads were filtered to remove reads with adaptor sequences, low-quality reads (Phred quality score <20 bp), and reads with a high percentage of unidentified nucleotides, using Perl script with G-language Genome Analysis Environment . De novo assembly of the clean reads was carried out using Trinity software (version: trinity/r2014-04-13p1) with default parameters and no reference sequence. The sequences resulting from the de novo Trinity assembly were called unigenes. In order to annotate unigenes, a BLASTX search against the UniProt database was conducted with an E-value cut-off of 1e−5. The following genomic databases were used for the taxonomic distribution of annotated components : plants (Chlamydomonas reinhardtii, http://www.ncbi.nlm.nih.gov/pubmed /17932292; Arabidopsis thaliana, https://www.arabidopsis.org/); animals (Drosophila melanogaster, http://flybase.org/; Caenorhabditis elegans, https://www.wormbase.org/); fungi (Saccharomyces cerevisiae, http://www.ensembl.org/index.html); kinetoplastids (Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, http://www.ncbi.nlm.nih.gov/). A venn diagram was drawn using Venny program (http://bioinfogp.cnb.csic.es/tools/venny/). The Blast2GO program  and Kyoto Encyclopedia of Genes and Genomes (KEGG) database  (http://www.genome.jp/kegg/) were used to identify the Gene ontology (GO) annotation and biological pathways in E.gracilis, respectively. Results of pathway enrichment were visualized using Pathway Projector .
Analysis of differentially expressed genes (DEG) in anaerbiosis E.gracilis cells
A differential gene expression analysis was performed using edgeR  (with FDR <0.05) on the reads that mapped with the bowtie2 software  to the unigenes assembled as described above For expression abundance estimation, the FPKM (Fragments Per Kilobase of exon per Million mapped fragments) values were computed using RSEM software  (http://www.ncbi.nlm.nih.gov/pubmed/21816040). Fold changes in differentially expressed genes in anaerobic-treated E.gracilis cells were calculated using the log2 ratio of FPKM. Based on the FPKM values, the pathway maps were generated using Pathway Projector. GO terms were extracted by matching UniProt-GOA associations with BLASTX search results (e-values <1e−5) and analyzed for statistically significant enrichment using GOstat for Biological Process terms (depth = 3; q-value ≤ 0.01; Benjamini correction for FDR) .
Availability of supporting data
The sequencing and transcriptome assembly data sets supporting the results of this study are deposited and available at the DDBJ Sequence Read Archive (DRA) with accession number SRP060591 and GDJR00000000.1 (GenBank), respectively. The data set supporting the results of this article is included within the article.
This work was supported by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST). This research was supported, in part, by research funds from the Yamagata Prefectural Government and Tsuruoka City, Japan.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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