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 posttranscriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.
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