Biosynthesis and Assembly of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185
Date
2019-07-08
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Degree Level
Doctoral
Abstract
Thraustochytrium is a marine protist producing a specific profile of nutritionally important fatty acids, including very long chain polyunsaturated fatty acids (VLCPUFAs) docosahexaenoic acid (DHA, 22:6n-3), even-chain saturated fatty acids (SFAs) palmitic acid (16:0), and odd-chain SFAs pentadecanoic acid (15:0). However, the mechanism of how these fatty acids are synthesized and assembled into the storage lipid triacylglycerol (TAG) is unclear.
Firstly, we reported sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Analysis of annotated genes from the genome revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of VLCPUFAs in this species. However, in the aerobic pathway, a key gene encoding stearoyl 9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. A genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT), an important enzyme in bridging two types of glycerolipids, diacylglycerol (DAG) and phosphatidylcholine (PC).
Secondly, a series of radiolabeled precursors were used to trace the biosynthetic process of different fatty acids in vivo and in vitro. When Thraustochytrium was fed with long chain fatty acid intermediates such as 14C-oleic acid, 14C-linoleic acid and 14C-α-linolenic acid, no VLCPUFAs were produced, indicating that the aerobic pathway for the biosynthesis of VLCPUFAs was not functional in Thraustochytrium. When fed with 14C-acetic acid, both SFAs and VLCPUFAs were labeled, and when fed with 14C-propionic acid, mainly SFAs were labeled. However, when fed with 14C-acetic acid in the presence of cerulenin, a type I FAS inhibitor, only VLCPUFAs were labeled, and when fed with 14C-propionic acid in the presence of cerulenin, neither SFAs nor VLCPUFAs were labeled. This result clearly indicates that the type I fatty acid synthase (FAS) in Thraustochytrium could use acetic acid and propionic acid as the primers to synthesize even-chain and odd-chain SFAs, respectively, and VLCPUFAs were synthesized by the PUFA synthase using acetic acid as the primer. The in vitro assay with 14C-malonyl-CoA in the presence of cerulenin showed that the crude protein of Thraustochytrium produced only VLCPUFAs, not SFAs, further confirming the role of the PUFA synthase in the biosynthesis of VLCPUFAs. These results have elucidated the biochemical mechanisms for the biosynthesis of all fatty acids in Thraustochytrium.
Thirdly, how freshly-synthesized fatty acids are incorporated into different glycerolipids was investigated. The glycerolipid profile of Thraustochytrium at log and stationary growth stages was analyzed by lipidomic tools, and then 14C-acetate and 14C-glycerol were used to trace the flux of fatty acids and backbone in glycerolipids. Lipidomic analysis showed that VLCPUFAs were mostly allocated to phosphatidylcholine (PC) and TAG. PC possessed a relatively stable profile of VLCPUFAs at the two growth stages, whereas TAG species with one or two VLCPUFAs were significantly increased at the stationary phase. Freshly-synthesized VLCPUFAs labeled by 14C-acetate were predominately incorporated into PC initially, while at the late time point of labeling, these fatty acids were mostly found in TAG. Positional analysis showed that PC had either one VLCPUFA at its sn-2 position (PC1) or two VLCPUFAs at both sn-1 and sn-2 positions (PC2), while TAG incorporated these fatty acids almost exclusively at the sn-2 position with similar stereospecific structure as PC1. Similarly, 14C-glycerol was more efficiently incorporated into PC1 than TAG initially, and at the late time point of labeling, it was mostly found in TAG, and DAG and PC1 shared a similar incorporation pattern. These results indicate that VLCPUFAs in TAG are mainly channeled from PC likely through diacylglycerol as the intermediate.
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Keywords
Thraustochytrium, very long chain polyunsaturated fatty acids, fatty acid biosynthesis, fatty acid assembly, triacylglycerol, phosphatidylcholine
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Food and Bioproduct Sciences
Program
Food Science