Study Of Important Nodes Involved In Triacylglycerol Synthesis And Accumulation Pathways In Phaeodactylum Tricornutum

Microalgae is a photosynthetic single-cell organism and contribute 40% of the world's primary productivity.Due to the high photosynthetic efficiency,easily cultivation,high biomass,no competition to farmland and easily genetic modification,microalgae are widely used in biofuels,medicines,foods and other fields.Among them,Phaeodactylum tricornutum is a single-cell model marine diatom and the genome information has been already published.P.tricornutum not only retain the characteristics of microalgae,but also contain high lipid content and polyunsaturated fatty acid composition.Besides,it can be genetically engineered to obtain high-lipid,high-PUFA engineered strains.The lipid droplet(LD)of microalgae is a monolayer organelle containing lipid.It has been found that LD is involved in oil membrane synthesis,lipid synthesis,lipid degradation,lipid flow and signal transduction.These biological processes are often regulated by LD proteins.Therefore,clarifying the structure and function of microalgal LD proteins can further understand the lipid accumulation process of microalgae and establish the firm foundation for microalgal biofuels industrialization.In this study,we selected the important genes involved in lipid and fatty acid synthesis in P.tricornutum to reveal the function of related genes by determination of physiological and biochemical parameters through cellular and molecular biology methods.The specific research findings are as follows:1.We identified 1?acyl?sn?glycerol?3?phosphate acyltransferase named AGPAT1 involved in triacylglycerol(TAG)biosynthesis pathway in P.tricornutum and found that AGPAT1 contains four conserved acyltransferase motifs I-IV.Immunoelectron microscopy result showed that AGPAT1 was localized in the chloroplast membrane of P.tricornutum.Overexpression of AGPAT1 reduced microalgal total carbohydrate content and soluble protein content but increased the lipid content of microalgae.AGPAT1 could coordinate the expression of other key genes in TAG synthetic pathway.In addition,AGPAT1 changed the fatty acid composition of microalgae,particularly the PUFA was increased.We observed the enlarged LD in the cytoplasm by overexpression of AGPAT1.Moreover,the ultrastructure result showed that plastoglobulus containing TAG were existed in chloroplast.The above results showed that overexpression of AGPAT1 can increase the TAG content and also reveal the diatom chloroplastid TAG synthetic pathway for the first time.2.Based on the research findings of Glycerol?3?phosphate acyltransferase(GPAT1)and AGPAT1 individually on lipid metabolism in P.tricornutum in our previous studies,we constructed two recombinant plasmids containing GPAT1 and AGPAT1 individually and introduced them into microalgae together.It was found that the co-overexpression of GPAT1 and AGPAT1 in microalgae promotes the accumulation of TAG.Growth and photosynthetic efficiency of transgenic microalgae were significantly enhanced in midlog phase,which might be caused by the additional synthesis of photosynthetic membranes in the chloroplast.In addition,we showed that chloroplast-localized GPAT1 and AGPAT1 utilized de novo synthesized fatty acids via the prokaryotic pathway for the chloroplastid TAG synthetic pathway for the first time.3.Microalgae containing high ratio of long chain polyunsaturated fatty acid(LCPUFA).This part reports a co-expression genetic engineering strategy by malonyl CoAacyl carrier protein transacylase(MCAT)and fatty acid desaturase 5b(D5b).The cooverexpressed strains promote the rapid accumulation of LC-PUFA.The contents of arachidonic acid(ARA)and docosahexaenoic acid(DHA)were as high as 18.98 ?g/mg and 9.15 ?g/mg,respectively.Importantly,by this strategy,we firstly accumulated the concentration of eicosapentaenoic acid(EPA)in microalgae up to 85.35 ?g/mg.In addition,we found that ARA and EPA were mainly accumulated in TAG of P.tricornutum,while DHA was mainly existed in phospholipids.Overall,the combined overexpression of two key enzymes resulted in high content of LC-PUFA without any affects on the growth of microalgae.4.To elucidate the proteins and regulatory mechanisms in LD of diatom,in this study,we identified LDP1,a gene encoding a lipid droplet associated protein in P.tricornutum and determined its function.LDP1 shares homology with another diatom Fistulifera sp.lipid droplet associated protein,DOAP1.Overexpression of LDP1 increased lipid content,lipid droplet size,and upregulated the transcript expression level of genes involved in TAG and fatty acid synthesis.Conversely,knock-down LDP1 by RNAi reduced lipid content and lipid droplet size.In addition,we isolated the lipid droplet of P.tricornutum treated with nitrogen-depletion and analyzed the lipid droplet protein components by mass spectrometry.We found that LDP1 was existed in the lipid droplet protein components with a high abundance.Importantly,we identified that LDP1 was localized in lipid droplet by using EYFP fluorescent protein.5.Patatin-like phospholipase domain-containing protein 3(PNPLA3)is associated with nonalcoholic fatty liver,which can promote lipid biosynthesis in the liver.We identified PNPLA3-ortholog gene in P.tricornutum.Overexpression of endogenous PNPLA3 promoted lipid content and also changed the fatty acid composition of microalgae;then we inserted the human Pnpla3I148 M into the genome of P.tricornutum.Human Pnpla3I148 M increased the lipid content and modify the preference of fatty acids in microalgae.In summary,these findings identified the microalgal PNPLA3,furthermore,overexpression of PNPLA3 can regulate the lipid biosynthesis.In addition,our data also showed that human Pnpla3I148 M can regulate microalgal lipid biosynthesis.These provide the important theoretical values for construction of engineered microalgal strains and elucidation of PNPLA3 function.Overall,this thesis firstly identified the existence of a chloroplast TAG synthesis and constitute mechanism in P.tricornutum and reported a novel lipid droplet associated protein named LDP1 involved in lipid synthesis and lipid droplet formation.We provided the multiple nodes simultaneous regulation straregy to promote the biosynthesis of lipid and PUFA and obtained the transformed microalgal strains with higher lipid and PUFA content.These results provided the deepened understanding of lipid metabolism mechanism in microalgae and also furnished the novel theoretical basis and ideals for high-value utilizations of microalgae.