Molecular mechanisms for biosynthesis and assembly of very long chain polyunsaturated fatty acids

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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.