The green microalga Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). Nevertheless, it remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis. To address these issues, we firstly determined the transcription start sites of 11 putative membrane-bound FAD-coding genes (CzFADs) and updated their gene models. Functional validation of these CzFADs in yeast and cyanobacterial cells revealed that seven are bona fide FAD enzymes with distinct substrates. Combining the validated functions and predicted subcellular compartments of CzFADs and the FA profiles of C. zofingiensis, the FA desaturation pathways in this alga were reconstructed. Furthermore, a multifaceted lipidomic analysis by systematically integrating thin-layer chromatography, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry techniques was conducted, unraveling profiles of polar membrane lipids in C. zofingiensis and major desaturation steps occurring in these lipids. By correlating transcriptional patterns of CzFAD genes and changes of lipids upon abiotic stress conditions, our results highlighted collaboration of CzFADs for C18 UFA synthesis and supported that both de novo FA synthesis and membrane lipid remodeling contributed C18 UFAs to triacylglycerol for storage. Taken together, our study for the first time elucidated the pathways of C18 FA desaturations and comprehensive profiles of polar membrane lipids in C. zofingiensis and shed light on collaboration of CzFADs for the synthesis and enrichment of C18 UFAs in triacylglycerol.

中文翻译：

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脂肪酸去饱和酶的表征揭示了产油藻 Chromochloris zofingiensis 中富含三酰基甘油的 C18 不饱和脂肪酸的应激诱导合成

绿色微藻 Chromochloris zofingiensis 能够产生高水平的富含 C18 不饱和脂肪酸 (UFA) 的三酰甘油。FA 去饱和度受 FA 去饱和酶 (FAD) 调节。然而，关于什么 FADs 参与 FA 去饱和以及这些 FADs 如何协同促进 C. zofingiensis 中三酰基甘油中 C18 UFA 的高丰度，仍然很大程度上未知。为了解决这些问题，我们首先确定了 11 个假定的膜结合 FAD 编码基因（CzFADs）的转录起始位点并更新了它们的基因模型。这些 CzFAD 在酵母和蓝藻细胞中的功能验证表明，有七种是具有不同底物的真正 FAD 酶。结合 CzFADs 的验证功能和预测的亚细胞区室以及 C. zofingiensis 的 FA 谱，重建了该藻类中的 FA 去饱和途径。此外，通过系统地整合薄层色谱、气相色谱-质谱和液相色谱-质谱技术，进行了多方面的脂质组学分析，揭示了 C. zofingiensis 中极性膜脂质的特征以及这些脂质中发生的主要去饱和步骤。通过将 CzFAD 基因的转录模式与脂质在非生物胁迫条件下的变化相关联，我们的研究结果强调了 CzFAD 在 C18 UFA 合成中的协作，并支持从头 FA 合成和膜脂重塑都将 C18 UFA 转化为甘油三酯进行储存。总之，我们的研究首次阐明了 C18 FA 去饱和的途径和 C.