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生物活性信号分子的前体。大量试验表明，膳食中的VLCPUFA可预防心血管疾病、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VLCPUFA的来源是野生鱼油和产油的VLCPUFA微生物。然而，由于养殖和提油成本高，来自海洋鱼类的石油被过度开发，来自含油微生物的石油价格昂贵。因此，利用来自VLCPUFA产生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一种潜在的替代方法。然而，迄今为止，在转基因油籽作物中产生所需的VLCPUFA水平和组成方面，这种尝试只取得了有限的成功。这意味着我们目前对本地微生物产生VLCPUFA的了解是有限的，从而阻碍了微生物VLCPUFA途径在油料植物中的有效实施。**VLCPUFA的从头生物合成只在某些类型的微生物中发生。已发现一些海洋微生物可以在储存油脂中产生大量VLCPUFA。在这些微生物中，VLCPUFAs的生物合成包括使用脱饱和酶和伸长酶引入双键并延伸现有脂肪酸的碳链以产生最终产品的有氧途径，或使用大型多结构域酶(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。这项研究的目标是阐明在Thraustochytrium sp 26185中生物合成VLCPUFA的分子机制，Thraustochytrium sp 100是一种能够在三酰甘油(TAG)中产生大量DHA的微生物。具体地说，我们将研究Thraustochytrium PUFA合成酶的新的催化机制，该酶是该物种生物合成DHA的主要酶，通过在异源宿主大肠杆菌中重组途径，结合定点突变和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，精确定位酰链中的顺式双键。此外，我们还将通过体内突变的放射性标记来研究两种不同的胆碱磷酸转移酶在促进磷脂和中性脂之间的酰基流动中的作用。通过跟踪标记底物通过中间体到最终甘油脂产物在Thraustochytrium突变株中的动力学，将揭示它们在VLCPUFA积累中的内在作用。这些目标的实现不仅有助于我们理解在天然微生物中通过厌氧途径生物合成VLCPUFAs的机制，而且也为在包括油料作物在内的异源系统中转基因生产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.