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

极长链多不饱和脂肪酸（VLC-PUFA或VLCPUFA）如花生四烯酸（20：4 n-6，ARA）、二十碳五烯酸（20：5 n-3，EPA）和二十二碳六烯酸（22：6 n-3，DHA）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。大量试验表明，膳食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。目前人类和动物的VLCPUFA来源是来自野生鱼类和产油VLCPUFA微生物的油。然而，海洋鱼类的油脂已经被过度开发，而产油微生物的油脂由于培养和油脂提取的高成本而昂贵。因此，使用来自VLCPUFA生产微生物的酶的油料作物的代谢工程被认为是供应这些脂肪酸的潜在替代方式。然而，就在转基因油籽作物中产生所需水平和组成的VLCPUFA而言，这些尝试迄今仅取得有限的成功。这意味着我们目前对天然微生物中VLCPUFA产生的理解是有限的，从而阻止了在油籽植物中有效实施微生物VLCPUFA途径。** VLCPUFA的从头生物合成仅发生在某些类型的微生物中。已经确定了许多海洋微生物可以在储存脂质中产生大量的VLCPUFA。这些微生物中VLCPUFA的生物合成涉及使用去饱和酶和延伸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的需氧途径，或使用大的多结构域酶（PUFA合酶）进行将初始丙二酰-CoA转化为VLCPUFA所需的所有反应的厌氧途径。本研究计划的目标是阐明破囊壶菌26185中VLCPUFA生物合成的分子机制，破囊壶菌26185是一种能够在三酰甘油（TAG）中产生大量DHA的微生物。具体而言，我们将调查新的催化机制破囊壶菌PUFA合酶，DHA的生物合成的主要酶的物种，精确定位顺式双键的酰基链中重建的途径，在异源宿主大肠杆菌结合定点诱变和体外测定的关键催化结构域（酮脂酰-ACP合酶和乙酰化酶）的PUFA合酶。此外，我们还将研究两种不同的胆碱磷酸转移酶在促进磷脂和中性脂质之间的酰基通量VLCPUFA积累过程中使用体内放射性标记的突变体的作用。通过以下的动力学标记底物通过中间体的最终甘油脂质产品的破囊壶菌突变体，其固有的作用VLCPUFA积累将被揭示。实现这些目标不仅有助于我们理解VLCPUFA在天然微生物中通过厌氧途径生物合成的潜在机制，而且还为在包括油料作物在内的异源系统中转基因生产VLCPUFA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.