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组成部分，也是生物活性信号分子的前体。大量科学报告表明，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，并提高眼睛、大脑和神经系统的表现。目前人类和动物VLCPUFA的来源是海鱼油和产油的VLCPUFA微生物。然而，来自海洋鱼类的石油被过度开发，来自微生物的石油由于微生物培养和石油提取的高成本而昂贵。因此，利用来自VLCPUFA产生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一种潜在的替代方法。然而，由于我们对VLCPUFA在天然微生物中的生物合成和组装了解不足，这种尝试迄今在转基因油籽作物中产生所需水平和组成的VLCPUFA方面只取得了有限的成功。VLCPUFAs的活性从头合成只在某些海洋微生物中发生。在这类微生物中VLCPUFAs的生物合成包括使用各种脱饱和酶和伸长酶引入双键并延伸中间酰链以产生最终产品的好氧途径或使用多不饱和脂肪酸合成酶(PUFA)的厌氧途径，PUFA合成酶是一种多功能巨型酶，用于执行将最初的丙二酰辅酶A前体转化为最终VLCPUFAs所需的所有反应。这项研究计划的目的是阐明在海洋原生生物Thraustochytrium中生物合成和组装VLCPUFA的分子机制。Thraustochytrium是一种海洋原生生物，可用于生产供人和动物食用的DHA。具体地说，我们将通过关键催化域(脱水酶和酮酰基合成酶)的体外分析和酶复合体的结构分析，研究PUFA合成酶(一种唯一负责原生体内DHA生物合成的酶)错综复杂地定位多个顺式双键的分子机制。此外，我们还将利用体外实验和体内甘油酯的酰基和骨架标记，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在卵磷脂转化为甘油二酯过程中的作用。实现这一目标不仅有助于我们了解VLCPUFA在天然微生物中的生物合成和组装机制，而且还将为有效实施微生物途径在油料作物中生产VLCPUFA提供新的策略。

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.