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

超长链多不饱和脂肪酸（VLCPUFAs）如二十二碳六烯酸（DHA, 22:6-n3）是哺乳动物细胞膜的重要成分和具有生物活性的信号分子的前体。许多科学报告表明，饮食中的VLCPUFAs可以预防心血管疾病、代谢综合征和炎症性疾病，并提高眼睛、大脑和神经系统的性能。目前人类和动物的vlcpufa来源是海洋鱼类的油和产油质vlcpufa的微生物。然而，海洋鱼类的油被过度开发，微生物的油由于微生物培养和提取成本高而价格昂贵。因此，利用产vlcpufa微生物的生物合成基因进行油籽植物代谢工程被认为是供应这些脂肪酸的潜在替代方法。然而，由于我们对天然微生物中VLCPUFAs的生物合成和组装的了解不足，迄今为止，这些尝试在转基因油籽作物中产生所需水平和组成的VLCPUFAs方面取得的成功有限。VLCPUFAs的活性新生生物合成仅发生在某些海洋微生物中。在这些微生物中，VLCPUFAs的生物合成包括好氧途径，利用各种去饱和酶和延长酶引入双键并延长中间酰基链以产生最终产物，或厌氧途径，利用多不饱和脂肪酸（PUFA）合成酶（一种多功能巨酶）进行将初始丙二酰辅酶a前体转化为最终VLCPUFAs所需的所有反应。本研究计划的目标是阐明在Thraustochytrium（一种商业上用于生产供人类和动物食用的DHA的海洋原生生物）中生物合成和组装VLCPUFAs的分子机制。具体来说，我们将研究PUFA合成酶（一种在原生生物中单独负责DHA生物合成的酶）的分子机制，通过对关键催化结构域（脱水酶和酮酰合成酶）的体外分析，以及对酶复合物的结构分析，来复杂地定位多个顺式双键。此外，我们还将研究VLCPUFAs在甘油脂中的通量，特别是两种关键酶，胆碱磷酸转移酶（CPT）和磷脂酶C，在磷脂酰胆碱到二酰基甘油的过程中所起的作用，这些酶在体外和体内对Thraustochytrium中的甘油脂进行酰基和骨干标记。这一目标的实现不仅有助于我们了解VLCPUFAs在天然微生物中生物合成和组装的机制，而且为油籽作物中VLCPUFAs生产的微生物途径的有效实施提供了新的策略。

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.