De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Fig. 1

Taxonomic distribution of annotated components by BLASTX search against genomic databases of plants, animals, fungi, and kinetoplastids. The genomic databases used are listed in Methods section. Significant matches were defined as those with e-values < 1e⁻⁵. The pie chart shows the percentage of no hits category of the BLAST results to NCBI nr database. The bar chart was added to visualize  gene numbers belonging to each category

Fig. 2

KEGG pathway analysis of assembled components in E.gracilis. The data exhibited are significantly enriched in the top 50 categories classified by KEGG BRITE hierarchy level 2 (http://www.genome.jp/kegg/kegg3b.html). a Illustration of the counts of the BRITE terms in all assembled components. b Analysis only for 800 up-regulated and 1280 down-regulated DEGs identified with FDR <0.05 in the comparison of aerobic and anaerobic states. The red and blue bars indicate the number of BRITE terms in up- and down-regulated genes, respectively

Fig. 3

Pathway map for paramylon and wax ester metabolism in E.gracilis. The enzymes involved in these pathways are provided. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose-1,6-bisphosphate; F2,6BP, fructose-2,6-bisphosphate; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 1,3BPG, 1,3-bisphosphoglycerate; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PEP, phosphoenolpyruvate; PNO, pyruvate:NADP⁺ oxidoreductase. The best e-values for individual genes are indicated in the top right corner of boxes shown enzyme names

Fig. 4

Representation of pathway maps for differential expressed genes in response to anaerobic treatment for 24 h. Given are values for FPKM ratios corresponding to components above the 2-fold change threshold. Blue (a) and red (b) colors highlight down- and up-regulated genes, respectively. Some genes related to pathways involving photosynthesis, nucleotide metabolism, TCA cycle, oxidative phosphorylation, fatty acid degradation, and a part of fatty acid biosynthesis showed lower expression in response to anaerobic treatment for 24 h. Zoomable interactive maps generated using Pathway Projector [39] are available online at: [Down-regulated pathways] http://ws.g-language.org/g4/main.cgi?diaAtabareaname=1&flag=00001, [Up-regulated pathways] http://ws.g-language.org/g4/main.cgi?diaAtabareaname=1&flag=00002