DataSheet_4_Lipid turnover and SQUAMOSA promoter-binding proteins mediate variation in fatty acid desaturation under early nitrogen deprivation revealed by lipidomic and transcriptomic analyses in Chlorella pyrenoidosa.docx

Nitrogen deprivation induces variations in fatty acid desaturation in microalgae, which determines the performance of biodiesel and the nutritional value of bioproducts. However, the detailed scenario and the underlying regulatory mechanism remain unclear. In this study, we attempt to outline these scenario and mechanisms by performing biochemical, lipidomic, and transcriptomic analyses in Chlorella pyrenoidosa and functional characterization of transcription factors in Yarrowia lipolytica. We found that early nitrogen deprivation dramatically reduced fatty acid desaturation without increasing lipid content. The contents of palmitic acid (16:0) and oleic acid (18:1) dramatically increased to 2.14 and 2.87 times that of nitrogen repletion on the second day, respectively. Lipidomic analysis showed the transfer of polyunsaturated fatty acids from phospholipids and glycolipids to triacylglycerols, and an increase in lipid species with 16:0 or 18:1 under nitrogen deprivation conditions. Upregulated stearoyl-ACP desaturase and oleyl-ACP thioesterase promoted the synthesis of 18:1, but restricted acetyl-CoA supply revealed that it was the intensive lipid turnover instead of an attenuated Kennedy pathway that played an important role in the variation in fatty acid composition under early nitrogen deprivation. Finally, two differentially expressed SQUAMOSA promoter-binding proteins (SBPs) were heterologously expressed in Y. lipolytica, demonstrating their role in promoting the accumulation of total fatty acid and the reduction in fatty acid desaturation. These results revealed the crucial role of lipid turnover and SBPs in determining fatty acid desaturation under early nitrogen deprivation, opening new avenues for the metabolic engineering of fatty acid desaturation in microalgae.

氮缺乏会诱导微藻的脂肪酸去饱和作用发生变化，这一过程决定了生物柴油的性能以及生物制品的营养价值。然而，其具体过程与潜在调控机制仍不明确。本研究以蛋白核小球藻（Chlorella pyrenoidosa）为材料开展生化、脂质组学与转录组学分析，并在解脂耶氏酵母（Yarrowia lipolytica）中对转录因子进行功能表征，以期阐明该过程与相关机制。研究发现，早期氮缺乏会显著降低脂肪酸去饱和水平，但并未提升脂质含量。培养至第2天时，棕榈酸（16:0）与油酸（18:1）的含量分别升至氮充足组的2.14倍与2.87倍。脂质组学分析显示，氮缺乏条件下，多不饱和脂肪酸从磷脂与糖脂转移至三酰甘油，且携带16:0或18:1的脂质物种丰度上升。上调的硬脂酰-ACP去饱和酶与油酰-ACP硫酯酶促进了18:1的合成，但乙酰辅酶A供应受限的结果表明，在早期氮缺乏条件下，驱动脂肪酸组成变化的并非减弱的肯尼迪途径（Kennedy pathway），而是剧烈的脂质周转过程。最后，我们将两个差异表达的SQUAMOSA启动子结合蛋白（SQUAMOSA promoter-binding proteins, SBPs）在解脂耶氏酵母中异源表达，证实了其可促进总脂肪酸积累并降低脂肪酸去饱和水平。本研究结果揭示了脂质周转与SBP类转录因子在早期氮缺乏条件下调控脂肪酸去饱和的关键作用，为微藻脂肪酸去饱和的代谢工程改造提供了新方向。