三角褐指藻LACS调控油脂合成的分子功能与调控机制研究

Free fatty acids exported from the plastid of eukaryotic microalge generally need to be re-activated to form fatty acyl-CoA thioesters by long-chain acyl CoA synthetases (LACS), and then participate in fatty acid-related metabolic pathways such as triacylglycerol (TAG) synthesis. Thus, LACS possibly plays an important role in lipid synthesis in the cell. The marine diatom Phaeodactylum tricornutum cells accumulate large amounts of TAGs upon nitrogen deprivation. However, TAG synthesis pathway and the mechanism by which TAG synthesis is regulated remain largely unknown at present. In the previous study, we have identified five members (ptACSL1-5) of the LACS protein family in P. tricornutum. In this proposal, we will use in vitro assay to determine substrate specificity of each ptACSL. We will use eGFP reporter gene fused with ptACSL, GFP self-assembly approach and immune-electron microscopy to determine subcellular localization of each ptACSL. For ptACSL proteins localized in the plastid, cytosol or lipid droplet, we will construct gene knockout mutants using TALEN-based gene editing technology. We will perform lipidomic analysis and profiling of acyl composition and content of the acyl-CoA pools in the generated mutants, which are grown under the condition of lipid accumulation. Based on the results from these experiments, we attempt to identify ptACSL(s) participating in regulation of lipid synthesis and reveal the related regulatory mechanism.

真核微藻质体输出的游离脂肪酸通常需要被长链酰基CoA合成酶(LACS)激活为脂肪酰基CoA的硫酯形式，然后参与脂肪酸相关的代谢途径例如甘油三酯(TAG)的合成，因此LACS可能在细胞油脂合成中起重要调控作用。海洋硅藻三角褐指藻细胞在氮源缺乏条件下积累大量TAG，然而目前关于TAG的合成途径及其调控机制仍未阐明。在前期研究中，我们鉴定出三角褐指藻LACS蛋白家族的5个成员（ptACSL1-5）。本项目采用体外实验确定每个ptACSL蛋白的底物特异性，采用eGFP报告基因融合方法、GFP自组装方法以及胶体金标记免疫电镜技术研究其亚细胞定位，采用TALEN基因编辑技术针对质体、细胞质或油滴定位的LACS蛋白构建其编码基因的敲除突变株，并研究油脂积累条件下突变株脂质组的变化以及酰基CoA库中脂肪酸组成和含量的变化规律，综合这些研究结果以鉴定出参与调控细胞油脂合成的LACS蛋白并阐明其调控机制。

长链酰基CoA合成酶(LACS)在油脂代谢中发挥多种重要作用。尽管细菌、酵母和植物中LACS功能有较为深入的研究，在单细胞产油微藻包括硅藻三角褐指藻中LACS酶调控油脂代谢的机制还不清楚。三角褐指藻基因组编码5个LACS酶(ptACSL1-5)，采用多重CRISPR/Cas9技术构建了lacs单基因敲除突变株，并确定了它们对于不同游离脂肪酸底物的偏好性以及亚细胞定位。ptACSL3定位于线粒体，敲除ptACSL3导致细胞在通气条件下生长变慢并且油脂含量减少；与野生型相比，ptACSL3突变株中两种半乳糖甘油脂和磷脂酰胆碱(PC)的脂肪酸组成发生改变，其中16:0和16:1的分布显著减少。ptACSL5定位于过氧化物酶体，敲除ptACSL5导致细胞生长速率降低，同时PC和甘油三酯的分子组成发生变化，表明其在调控酰基CoA组成用于油脂合成中发挥作用。本项目的研究不仅表明多重CRISPR/Cas9技术在构建大片段缺失基因敲除突变株中有很好的潜力，同时也为认识LACS酶在产油微藻油脂代谢中的功能提供基础。