Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids
Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids
复制标题
对硅藻三角褐指藻中长链酰基辅酶A合成酶家族的多重CRISPR/Cas9编辑揭示线粒体ptACSL3参与储存脂质的合成
DOI：
10.1111/nph.17911
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发表时间：
2021-12-20
期刊：
影响因子：
9.4
通讯作者：
Gong, Yangmin
中科院分区：
文献类型：
--
作者：
Hao, Xiahui;Chen, Wenchao;Gong, Yangmin
长链酰基辅酶A合成酶（拉克）在脂质代谢中发挥着多种重要作用。虽然它们的功能已经在细菌、酵母和植物中得到很好的确立，但拉克同工酶调节单细胞产油微藻（包括硅藻三角褐指藻）中的脂质代谢的机制在很大程度上仍然未知。在三角褐指藻中，一个由五个基因（ptACSL 1-ptACSL 5）组成的家族编码拉克活性。我们使用多重CRISPR/Cas9方法产生单个拉克敲除/敲低突变体，并确定它们对不同脂肪酸（FA）和亚细胞定位的底物特异性。ptACSL 3定位于线粒体中，当细胞用空气鼓泡时，其破坏导致生长受损和三酰甘油（TAG）含量降低。ptACSL 3突变体显示改变FA配置文件中的两个半乳糖甘油酯和磷脂酰胆碱（PC）的分布显着减少16：0和16：1。ptACSL 5位于过氧化物酶体中，其敲低导致生长速率降低和PC和TAG分子种类改变，表明其在控制脂质合成的酰基辅酶A组成中的作用。我们的工作证明了使用多重CRISPR/Cas9产生具有大片段缺失突变的基因敲除突变体的潜力，并提供了对产油微藻中拉克同工酶在脂质代谢中的功能的深入了解。
Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. We generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.