Molecular mechanisms for biosynthesis and assembly of very long chain polyunsaturated fatty acids

极长链多不饱和脂肪酸（VLCPUFA）如二十二碳六烯酸（DHA，22：6-n3）是哺乳动物细胞膜的必需组分和生物活性信号分子的前体。许多科学报告表明，膳食中的VLCPUFA可以预防心血管疾病，代谢综合征和炎症性疾病，并增强眼睛，大脑和神经系统的性能。目前人类和动物的VLCPUFA来源是来自海洋鱼类和产油VLCPUFA微生物的油。然而，来自海洋鱼类的油已经被过度开发，并且由于微生物培养和油提取的高成本，来自微生物的油是昂贵的。因此，使用产VLCPUFA微生物的生物合成基因对油料植物进行代谢工程被认为是供应这些脂肪酸的潜在替代方式。然而，由于我们对天然微生物中VLCPUFA的生物合成和组装的了解不足，这些尝试迄今为止在转基因油料作物中产生所需水平和组成的VLCPUFA方面仅取得了有限的成功。 VLCPUFA的主动从头生物合成仅发生在某些海洋微生物中。VLCPUFA在此类微生物中的生物合成涉及使用各种去饱和酶和延伸酶引入双键并延伸中间酰基链以产生最终产物的需氧途径，或使用多不饱和脂肪酸（PUFA）合酶（多功能巨酶）进行初始丙二酰辅酶A前体转化为最终VLCPUFA所需的所有反应的厌氧途径。这项研究计划的目标是阐明破囊壶菌（Thraustochytrium）中VLCPUFA生物合成和组装的分子机制，破囊壶菌是一种海洋原生生物，用于生产DHA供人类和动物食用。具体而言，我们将调查的PUFA合酶，一种酶，只负责DHA的生物合成的原生生物，复杂的定位多个顺式双键使用关键催化结构域（脱氢酶和酮脂酰合酶）的体外测定的分子机制，和酶复合物的结构分析。此外，我们还将调查的流量VLCPUFAs的甘油脂，特别是两个关键酶，胆碱磷酸转移酶（CPT）和磷脂酶C，在从磷脂酰胆碱的过程中的作用，使用体外测定和体内酰基和骨干标记的甘油脂在破囊壶菌。实现这一目标不仅有助于我们理解VLCPUFA在天然微生物中的生物合成和组装机制，而且还为有效实施油料作物中VLCPUFA生产的微生物途径提供了新的策略。

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6-n3) are essential components of cell membrane and precursors for biologically active signaling molecules in mammals. Numerous scientific reports have shown that dietary VLCPUFAs can provide protection against cardiovascular diseases, metabolic syndrome and inflammatory disorders, and enhance the performance of eyes, brain and nervous systems. The current sources of VLCPUFAs for humans and animals are oils from marine fish and oleaginous VLCPUFA-producing microorganisms. However, oil from oceanic fish has been over-exploited and oil from the microbes is expensive due to the high cost in microbial culture and oil extraction. Metabolic engineering of oilseed plants using the biosynthetic genes from VLCPUFA-producing microorganisms has thus been considered as a potential alternative way to supply these fatty acids. However, such attempts have so far achieved only limited success in terms of producing the desirable level and composition of VLCPUFAs in transgenic oilseed crops due to our inadequate understanding of the biosynthesis and assembly of VLCPUFAs in the native microorganisms. Active de novo biosynthesis of VLCPUFAs occurs only in certain marine microorganisms. Biosynthesis of VLCPUFAs in such microbes involves either an aerobic pathway using a variety of desaturases and elongases to introduce double bonds and extend intermediate acyl chains for producing the final products or an anaerobic pathway using a polyunsaturated fatty acid (PUFA) synthase, a multi-functional mega-enzyme to carry out all reactions required for the conversion of initial malonyl-CoA precursor to final VLCPUFAs. The goal of this research proposal is to elucidate molecular mechanisms for the biosynthesis and assembly of VLCPUFAs in Thraustochytrium, a marine protist commercially utilized for the production of DHA for human and animal consumption. Specifically, we will investigate the molecular mechanism of the PUFA synthase, an enzyme solely responsible for the biosynthesis of DHA in the protist, for intricately positioning multiple cis-double bonds using in vitro assays of key catalytic domains (dehydratase and ketoacyl synthase), and structural analysis of the enzyme complex. In addition, we will also investigate the flux of VLCPUFAs in glycerolipids, particularly the roles of two critical enzymes, cholinephosphotransferase (CPT) and phospholipase C, in the process from phosphatidylcholine to diacylglycerol using in vitro assays and in vivo acyl and backbone labeling of glycerolipids in Thraustochytrium. Achieving this goal will not only contribute to our understanding of the mechanisms underlying the biosynthesis and assembly of VLCPUFAs in the native microbe, but also provide new strategies for the effective implementation of the microbial pathways for the production of VLCPUFAs in oilseed crops.