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.

在厌氧条件下，Euglena gracilis通过线粒体脂肪酸从称为对mylon的储存多糖中合成大量蜡酯，从而生成ATP。线粒体中的反式-2-烯酰基辅酶A还原酶（TERs）被认为在此过程中起关键作用，因为这些酶催化短链长度辅酶A底物（如巴豆酰辅酶A）的还原。TER酶（EgTER1）先前已被鉴定并进行了酶学鉴定。然而，其生理学意义仍有待通过遗传分析进行评估。我们在这里生成了EgTER1-nockdown Euglena细胞，其中巴豆酰辅酶A总还原酶活性降低至对照值的10％。值得注意的是，击倒细胞表现出严重的漂白表型，缺乏叶绿素和糖脂，但在异养条件下（补充葡萄糖）正常生长。而且，敲除细胞在转移到厌氧条件之前和之后比对照细胞积累了更多的蜡酯，这伴随着较大的代谢组学变化。此外，我们未能发现蜡酯生产中其他潜在的TER基因的任何贡献。我们的发现提出了EgTER1在绿化过程中的新作用，并证明该酶可在厌氧条件下生产蜡酯。