A Preliminary Study On The Function Of Phaeodactylum Tricornutum LACS Enzymes In Fatty Acid Metabolism

Recently,the importance of long-chain polyunsaturated fatty acids?LC-PUFAs?for human health has receivedincreasing attention.At present,the major source of LC-PUFAs for humans is deep-sea fish oils.However,fish oils have the risk of resource shortage and environmental pollution,so it is vital to seek new alternative sources.Marine microalgae are able to synthesize LC-PUFAs and are the primary producers of marine LC-PUFAs.The study on LC-PUFA biosynthetic and metabolic pathways in marine microalgae is of great significance for the development of alternative sources of LC-PUFAs.Long-chain acyl-CoA synthetases?LACS?plays critical roles in fatty acid metabolism.Free fatty acids must be activated by LACS to form their corresponding CoA esters that are used for subsequent metabolic pathways,such as carbon chain elongation,triacylglycerol synthesis,glycolipid synthesis,phospholipid synthesis,beta oxidation,etc.Uncovering the functions of LACS enzymes in lipid metabolism in diatoms is helpful for understanding of LC-PUFA metabolic pathway.In this study,the marine microalga Phaeodactylum tricornutum rich in LC-PUFAs was used to study the biochemical properties of five long-chain acyl-CoA synthetase s?ptACSL1-5?using molecular biology and biochemical methods.The main findings are as follows:?1?Genes encoding ptACSL1-4 were amplified by reverse transcription PCR.Expression plasmids of ptACSL1-4 fused with eGFP were constructed and transformed into Phaeodactylum tricornutum.Subcellular localization of each ptACSL:GFP fusion protein was determined by observing GFP fluorescence.The results show that:ptACSL1,ptACSL2 and ptACSL3 are localized in the plastid;ptACSL4 is localized in both plasma membrane and the cytosol,and ptACSL5 is localized in the peroxisome.The specific location of ptACSL1 in the plastid was further determined by GFP self-assembly method.The N-terminal transmembrane region of ptACSL1 protein was localized on the chloroplast ER membrane,and the C-terminus was localized in the cytosol.?2?Substrate specificities of ptACSL1-5 proteins were measured by enzyme activity in vitro.The expression plasmids of ptACSL1-5 protein fused with 6×His tag at the C-terminus were constructed,respectively,and transformed into Escherichia coli for heterologous expression.Recombinant proteins of ptACSL:6×His were purified by affinity chromatography.The activity of each recombinant protein was determined by NADH depletion method with different free fatty acids as substrates in in vitro enzyme reaction system.The results show that when C18:1 was used as the substrate,the enzyme activities of all five ptACSL proteins were relatively high?175.50-330.38 nmol/min/mg?;The enzyme activities of ptACSL1 and ptACSL2 could be detected in the reaction system using different free fatty acids as substrates;When C20:5 was used as the substrate,the enzyme activities of ptACSL1 and ptACSL4 were two orders of magnitude higher than other substrates,reaching 3135.05 nmol/min/mg and 2388.8 nmol/min/mg,respectively.?3?Western blot was used to determine the expression patterns of ptACSL1-5 proteins under the conditions of nitrogen deficiency and elevating CO₂ concentration?air,1%,4%,10%?.Within 72 hours of nitrogen deficiency,the protein expression of ptACSL1,ptACSL3 and ptACSL4 was continuously up-regulated,while ptACSL2 expression was down-regulated.With the increase of CO₂ concentration,the expression of ptACSL1,ptACSL2 and ptACSL4 protein decreased;with the increase of CO₂concentration from air to 4%,the expression of ptACSL3 increased greatly,and then be inhibited when the CO₂ concentration was further increased to 10%.?4?CRISPR/Cas9 technology was used to construct ptACSL1-5 gene knockout mutants.Six sgRNAs were designed to target each ptACSL gene,and the corresponding CRISPR/cas9 knockout plasmids were constructed.Genomic DNAs from the transformants were used as templates to detect mutation and sequence the gRNA targeting sites.The results show that mutation types are all fragment deletions with the deletion size of 67-733bp,and single gene mutation rate is about 42-60%.About 3-5mutant strains were finally selected for each ptACSL gene for subsequent analysis.In conclusion,subcellular localization,substrate specificities and expression patterns of five ptACSL proteins from Phaeodactylum tricornutum were characterized,and knockout mutants for each ptACSL gene were constructed,which laid a foundation for future elucidating of the molecular regulatory functions of ptACSL1-5 in fatty acid metabolism in this model diatom.