Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, Japan.
PLoS One. 2019 Jan 16;14(1):e0210755. doi: 10.1371/journal.pone.0210755. eCollection 2019.
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，衣藻通过从称为副淀粉的储存多糖进行线粒体脂肪酸合成来大量产生蜡酯，以产生 ATP。线粒体中的反式 2-烯酰-CoA 还原酶（TERs）被认为在这个过程中起着关键作用，因为这些酶催化短链 CoA 底物（如巴豆酰-CoA）的还原。先前已经鉴定并酶学表征了一种 TER 酶（EgTER1）；然而，其生理意义仍需要通过遗传分析来评估。我们在此生成了 EgTER1 敲低衣藻细胞，其中总巴豆酰-CoA 还原酶活性降低至对照值的 10%。值得注意的是，敲低细胞表现出严重的白化表型，缺乏叶绿素和糖脂，但在异养条件（葡萄糖补充）下正常生长。此外，敲低细胞在转移到厌氧条件前后积累的蜡酯量比对照细胞多得多，这伴随着大量的代谢组学变化。此外，我们未能发现其他潜在的 TER 基因在蜡酯生产中的任何作用。我们的发现提出了 EgTER1 在绿色化过程中的新作用，并表明该酶在厌氧条件下对于蜡酯生产是可有可无的。
