Molecular analysis of mechanisms underlying biosynthesis of very long chain polyunsaturated fatty acids

超长链多不饱和脂肪酸（VLC-PUFAs或VLCPUFAs）如花生四烯酸（20:4n-6， ARA）、二十碳五烯酸（20:5n-3， EPA）和二十二碳六烯酸（22:6n-3， DHA）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。大量试验表明，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，并增强眼睛、大脑和神经系统的功能。目前人类和动物的vlcpufa来源是野生鱼类的油和产油质vlcpufa的微生物。然而，海洋鱼类的油被过度开发，而产油微生物的油由于培养和提取成本高而价格昂贵。因此，利用产vlcpufa微生物的酶进行油籽作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，就在转基因油籽作物中产生理想的VLCPUFAs水平和组成而言，这些尝试迄今仅取得了有限的成功。这意味着我们目前对原生微生物中VLCPUFA产生的理解是有限的，从而阻碍了油籽植物中微生物VLCPUFA途径的有效实施。** VLCPUFAs的从头生物合成仅发生在某些类型的微生物中。许多海洋微生物已经被确定可以在储存脂质中产生大量的VLCPUFAs。在这些微生物中，VLCPUFAs的生物合成既包括好氧途径，利用去饱和酶和延长酶引入双键并延长预先存在的脂肪酸的碳链以产生最终产物，也包括利用大型多结构域酶（PUFA合成酶）进行将初始丙二酰辅酶a转化为VLCPUFAs所需的所有反应的厌氧途径。本研究计划的目标是阐明Thraustochytrium sp 26185中VLCPUFAs生物合成的分子机制，Thraustochytrium sp 26185是一种能够在三酰基甘油（TAGs）中产生大量DHA的微生物。具体来说，我们将研究Thraustochytrium PUFA合成酶（该物种生物合成DHA的主要酶）的新催化机制，通过在异源宿主大肠杆菌中重组途径，结合位点定向诱变和PUFA合成酶的关键催化结构域（酮酰基- acp合成酶和脱水酶）的体外分析，精确定位酰基链上的顺式双键。此外，我们还将研究两种不同的胆碱磷酸转移酶在VLCPUFAs积累过程中促进磷脂和中性脂之间酰基流动的作用，并对其突变体进行体内放射性标记。通过跟踪标记底物在Thraustochytrium突变体中从中间体到最终甘油脂产物的动力学，将揭示它们在VLCPUFA积累中的本地作用。这些目标的实现不仅有助于我们理解原生微生物通过厌氧途径生物合成VLCPUFAs的机制，而且为在包括油籽作物在内的异种系统中转基因生产VLCPUFAs提供了潜在的新策略。

Very long chain-polyunsaturated fatty acids (VLC-PUFAs or VLCPUFAs) such as arachidonic acid (20:4n-6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are essential components of cell membrane and precursors for biologically active signaling molecules in mammals. Numerous trials have shown that dietary VLCPUFAs 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 wild fish and oleaginous VLCPUFA-producing microorganisms. However, oil from marine fish has been over-exploited and oil from oleaginous microbes is expensive due to the high cost in culturing and oil extraction. Metabolic engineering of oilseed crops using enzymes 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. This implies that our current understanding of the production of VLCPUFAs in native microorganisms is limited, thereby preventing effective implementation of microbial VLCPUFA pathways in oilseed plants. **De novo biosynthesis of VLCPUFAs only occurs in certain types of microorganisms. A number of marine microbes have been identified that can produce substantial amounts of VLCPUFAs in the storage lipids. The biosynthesis of VLCPUFAs in these microbes involves either an aerobic pathway using desaturases and elongases to introduce double bonds and extend carbon chains of pre-existing fatty acids for producing the final products or an anaerobic pathway using a large multi-domain enzyme (PUFA synthase) to carry out all reactions required for conversion of initial malonyl-CoA to VLCPUFAs. The goal of this research program is to elucidate molecular mechanisms for the biosynthesis of VLCPUFAs in Thraustochytrium sp 26185, a microbe capable of producing a substantial amount of DHA in triacylglycerols (TAGs). Specifically, we will investigate novel catalytic mechanisms of Thraustochytrium PUFA synthase, the primary enzyme for the biosynthesis of DHA in the species, for precisely positioning cis-double bonds in the acyl chain by reconstituting the pathway in a heterologous host Escherichia coli in combination with site-directed mutagenesis and the in vitro assays of key catalytic domains (ketoacyl-ACP synthase and dehydratase) of the PUFA synthase. In addition, we will also investigate roles of two different cholinephosphotransferases in facilitating acyl flux between phospholipids and neutral lipids during accumulation of VLCPUFAs using in vivo radioactive labeling of their mutants. By following the kinetics of the labeled substrates through intermediates to the final glycerolipid products in the Thraustochytrium mutants, their indigenous roles in VLCPUFA accumulation will be revealed. Achieving these goals would not only contribute to our understanding of the mechanisms underlying the biosynthesis of VLCPUFAs via the anaerobic pathway in the native microbes, but also provide potential new strategies for transgenic production of VLCPUFAs in heterologous systems including oilseed crops.