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

点击翻译按钮获取中文摘要

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.