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.
Highlights
Euglena gracilis is a single-celled eukaryotic alga containing chloroplasts with photosynthetic activity
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
Semiquantitative Reverse transcription (RT)-polymerase chain reaction (PCR) and protein blotting confirmed that EgTER1 expression was markedly reduced in the knockdown cells compared to that in the mock control (Fig 2A and 2B)
Summary
Euglena gracilis is a single-celled eukaryotic alga containing chloroplasts with photosynthetic activity. Photosynthetic products are stored as polysaccharides termed paramylon, which consist of only liner β-1,3-glucan, in Euglena grown under aerobic conditions [1,2]. Paramylon content often exceeds 50% of the dry weight of the cell [3,4]. Mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena collection and analysis, decision to publish, or preparation of the manuscript
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