Dataset Information

Succinate and Lactate Production from Euglena gracilis during Dark, Anaerobic Conditions.

ABSTRACT: Euglena gracilis is a eukaryotic, unicellular phytoflagellate that has been widely studied in basic science and applied science. Under dark, anaerobic conditions, the cells of E. gracilis produce a wax ester that can be converted into biofuel. Here, we demonstrate that under dark, anaerobic conditions, E. gracilis excretes organic acids, such as succinate and lactate, which are bulk chemicals used in the production of bioplastics. The levels of succinate were altered by changes in the medium and temperature during dark, anaerobic incubation. Succinate production was enhanced when cells were incubated in CM medium in the presence of NaHCO3. Excretion of lactate was minimal in the absence of external carbon sources, but lactate was produced in the presence of glucose during dark, anaerobic incubation. E. gracilis predominantly produced L-lactate; however, the percentage of D-lactate increased to 28.4% in CM medium at 30°C. Finally, we used a commercial strain of E. gracilis for succinate production and found that nitrogen-starved cells, incubated under dark, anaerobic conditions, produced 869.6 mg/L succinate over a 3-day incubation period, which was 70-fold higher than the amount produced by nitrogen-replete cells. This is the first study to demonstrate organic acid excretion by E. gracilis cells and to reveal novel aspects of primary carbon metabolism in this organism.

SUBMITTER: Tomita Y

PROVIDER: S-EPMC5174102 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Succinate and Lactate Production from Euglena gracilis during Dark, Anaerobic Conditions.

Tomita Yuko Y Yoshioka Kazumasa K Iijima Hiroko H Nakashima Ayaka A Iwata Osamu O Suzuki Kengo K Hasunuma Tomohisa T Kondo Akihiko A Hirai Masami Yokota MY Osanai Takashi T

Frontiers in microbiology 20161221

Euglena gracilis is a eukaryotic, unicellular phytoflagellate that has been widely studied in basic science and applied science. Under dark, anaerobic conditions, the cells of E. gracilis produce a wax ester that can be converted into biofuel. Here, we demonstrate that under dark, anaerobic conditions, E. gracilis excretes organic acids, such as succinate and lactate, which are bulk chemicals used in the production of bioplastics. The levels of succinate were altered by chan ...[more]

PMID: 28066371

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Project description:BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Project description:The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77-0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.

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Succinate and Lactate Production from Euglena gracilis during Dark, Anaerobic Conditions.

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Succinate and Lactate Production from <i>Euglena gracilis</i> during Dark, Anaerobic Conditions.

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