A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions

Abstract

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

Fig 1. Overview of the pathway for wax ester synthesis in Euglena gracilis.

Following glycolysis in the cytosol, fatty acids are synthesized by the reverse reaction of β-oxidation as highlighted in the right side of the figure. trans-Enoyl-CoA reductase plays a key role in the fatty acid synthesis, rather than acyl-CoA dehydrogenase.

Fig 2. Effect of EgTER1-knockdown on cell growth and chlororphyll content in heterotrophically grown cells.

(a) RT-PCR for verification of EgTER1 gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. Mock cells were electroporated without dsRNA. (b) Immuno detection of TER1 protein. (c) Comparison of TER activity between mock control and KD-ter1 cells. Euglena cells grown to stationary phase were collected for TER activity measurement as described in the Material and Method section. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. Values represent the means ± SD of three independent experiments. (d) Growth curve of mock control (blue) and KD-ter1 (orange) cells. The cultures were incubated in the heterotrophic KH medium. (e) Green color-less phenotype of KD-ter1 cells. The picture shows a representative culture after 7 days growth in the KH medium. (f) Influence of EgTER1-knockdown on chlorophyll content. An asterisk denotes statistically significant differences (**P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments. (g) Effect of gene expression levels of other TER orthologs on EgTER1-knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced.

Fig 3. Effect of EgTER1-knockdown on some key compounds in wax ester fermentaion.

(a) Influence of EgTER1-knockdown on myristyl myristate production under anaerobic conditions. Euglena cells grown to stationary phase were anaerobically treated for 24 h and then collected for wax ester measurement as described in the Material and Method section. The inset shows the results for 0 h in detail. An asterisk denotes statistically significant differences (*P<0.05, **P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments. (b) Paramylon contents in EgTER1-knockdown cells. Cells grown to stationary phase were anaerobically treated for 24 h and collected for paramylon measurement. Values represents the means ± SD of three independent experiments. (c) Fatty acid content. Cells grown to stationary phase were anaerobically treated for 24 h and collected for fatty acids measurement. Values represent the means ± SD of three independent experiments. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. (d) TER activity prior to and following anaerobic treatment. Values represent the means ± SD of three independent experiments.

Fig 4

Concentrations of some key metabolites related to the glycolysis (a), TCA cycle (c), storage lipid (c) and membrane lipid (d) metabolism. Cells grown to stationary phase were anaerobically treated for 24 h and collected for metabolome analysis. Concentrations and relative amounts of each metabolite in the mock control and EgTER1-knockdown cells are shown. An asterisk denotes statistically significant differences (*P<0.05, **P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments.

Fig 5. Effect of gene knockdown for other TER orthologs on wax ester production.

(a) RT-PCR for verification of each possible TER gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. (b) Chlorophyll content in possible TER gene knockdown cells. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. Values represent the means ± SD of three independent experiments. (c) Wax ester production under anaerobic conditions. Euglena cells grown to stationary phase were anaerobically treated for 24 h and collected for myristyl myristate measurement as described in the Material and Method section. Values represent the means ± SD of three independent experiments.