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oai:www.repository.cam.ac.uk:1810/3047532020-04-29T06:47:06Zcom_1810_223857com_1810_256062col_1810_223858

Investigating mechanisms of triacylglycerol synthesis induced by nitrogen depletion in the diatom Phaeodactylum tricornutum and characterising its diacylglycerol acyltransferases and phospholipid: diacylglycerol acyl transferase 10.17863/CAM.51835 Bower, Iain David 0000000266392145 Smith, Alison The ability of the diatom Phaeodactylum tricornutum (P. tricornutum) to accumulate triacylglycerol (TAG) and synthesise the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) has led to interest in their development for biofuels and human nutrition. Nitrogen depletion is a well-studied experimental system for producing high levels of TAG in P. tricornutum. Identication of the mechanisms involved in TAG synthesis holds promise for increasing TAG yields. Numerous studies of nitrogen depletion have been carried out that combine physiological and transcriptomic or proteomic measurements. These studies provide insight into global and local metabolic responses but experimental setups varied. To identify a consistent response, experimental conditions and physiological changes were compiled and transcripts and proteins were functionally grouped in a systematic review. Comprehensive analysis of lipid metabolism and autophagy genes was undertaken. Analysis of regulated genes supports involvement of central pathways and lipid metabolism in TAG accumulation: acetyl-CoA synthesis may increase while FA synthesis, the Kennedy pathway, malonyl-CoA/ACP transacylase, plastidial desaturase and FA transport enzymes were upregulated. Desaturases, elongases, Lands cycle enzymes and long chain acyl-CoA synthases are targets for overexpression to increase EPA production during nitrogen depletion. Several lipidomic experiments have highlighted the potential for TAG synthesis from chloroplast lipids, particularly monogalactosyl diacylglycerol (MGDG), during disassembly of thylakoid membranes under N-depletion. Identifying MGDG derived TAG accumulation may yield enzymes that target EPA to TAG and thereby enable use of developed extraction processes. Some archaeplastida also synthesise TAG from MGDG in nitrogen depletion and freezing stress. A similar response in diatoms would demonstrate this metabolic feature in diverse photosynthetic lineages. In this thesis, the fatty acid synthase inhibitor cerulenin was used to probe TAG accumulation during N-depletion. Neutral lipid staining data support the possibility of an MGDG to TAG route but were not conclusive. Concurrently, candidate enzymes were identied based on conserved sequence domains and experimentally characterised genes from Chlamydomonas reinhardtii (C. reinhardtii ) and Arabidopsis thaliana (A. thaliana). P. tricornutum was subsequently transformed with acyl hydrolase:YFP constructs. Confocal microscopy supported locations of two acyl hydrolases: in the endoplasmic reticulum (ER) and chloroplast ER for Phatr3 J44028 and in the ER or cytoplasm for Phatr3 J41624. P. tricornutum encodes multiple isoforms of the TAG synthesis enzymes diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Previous studies supported functionality of multiple PtDGATs and dierential regulation and predicted locations suggest non-redundancy. To investigate their role, overexpression of multiple DGATs and an N-terminal truncated PDAT was induced under nitrogen depletion. Total lipid extraction was carried out and species and FA composition of TAG was measured using tandem mass spectrometry. Total TAG was not altered. Overexpression of DGAT2B decreased the proportion of the 48:2 (carbon atoms:double bonds) TAG species and increased 50:2 and 50:3 while palmitic and oleic acid formed an increased and decreased proportion of TAG respectively. Overexpression of PDAT decreased the 48:1 and 48:2 TAG species, decreased the proportion of TAG composed of palmitic acid and increased the amount composed of EPA. Knowledge of typical TAG species composition in P. tricornutum supported respective preferences for incorporation of 18 carbon acyl-CoAs and EPA by DGAT2B and PDAT and their use of C16/C16 diacylglycerols. University of Cambridge 2021-01-30 Thesis Doctoral Doctor of Philosophy (PhD) en Sponsored as part of a BBSRC sLOLA grant https://www.repository.cam.ac.uk/handle/1810/304753 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fd8bbd9a-fbdf-4642-8a87-85f0eb97fc16/download fdd8a7fc9484daa2cfcf9d0f6d2017b5 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b374a3a0-40e7-4b54-9886-1a5a493ff13f/download 87eda9de84448d1f82354d60eee3eb5f https://creativecommons.org/licenses/by-nc-sa/4.0/ algae alga tag triacylglycerol dgat pdat phaeodactylum tricornutum nitrogen depletion nitrogen starvation diatom transcriptomic oil remodelling mgdg acyl transferase