Cloning And Functional Characterization Of Long-chain Acyl-CoA Synthetase 1 And Long-chain Acyl-CoA Synthetase 9 From The Mesocarp Of African Oil Palm(Elaeis Guineensis Jacq.)

As the most productive world oil crop,oil palm is of very vital source of palm oil that is derived from the fruit mesocarp where it can comprise up to 90%of the dry weight.Previously,many studies revealed that LACS is one of key enzymes play versatile roles on fatty acids metabolism pathways.It was implied that EgLACS1 and EgLACS9 could exert positive function on oil biosynthesis in oil palm.All of them contain a highly conserved signature domain required for AMP binding and adenylate formation.Except for that,the ACS signature motif and LACS-specific linker domain are both present in these three variants proteins.EgLACS1 with AtLACS1,BnLACS1 and AhLACS1,belong to Clade I,which was well known participated in plant's very-long-chain fatty acid(VLCFA)synthesis in wax and cutin.EgLACS9 AtLACS9,BnLACS9 and GmLACS9 belong to Clade I,which was well knownparticipated in the transport from plastid to endoplasmic reticulum.EgLACS 1 and EgLACS9 can encode active long acyl-CoA synthetase and capable of compensation yeast YB525,which lacks both FAA1 and FAA4 and was considered a well-characterized model system to identify candidate ACSL genes.Additionally,the transformants of EgLACS1 and EgLACS9 not only provide an efficient way to verify the activity of LACS enzymes but also supply a path to elucidate the preferred substrate.However,there is no significant preference in utilization of all of these fatty acids substrates,neither EgLACSl or EgLACS9.To further investigate the import function of EgLACS 1,C1-BODIPY-C12,a fluorescent C18 free fatty acid analogue,was employed in YB525 medium.This experiment was closely mirrored the growth complementation results,suggesting that the ability of EgLACS1 and EgLACS9 in LACS activity and uptake of fatty acids.In yeast and plants,?-oxidation occurs exclusively in peroxisomes.n this study,the important composition of fatty acids were significantly reduced in EgLACS1-V1oxEgLACS1-V2ox and EgLACS9 YB525 straincompared with the control carrying empty vector,whereas in which such fatty acids species exhibits significantly increased when compared with YB332 WT.The loss of LACS activity in YB525 mutant strain causes them unable to activate fatty acids C16:0-C18:1 and participate in the catabolism of fatty acids through?-oxidationpathways,and render the retention of fatty acids in intracellular cytoplasm.According all of above results,a proposed model illustrates that EgLACS1 and EgLACS9are a peripheral protein;ACS encoded by such EgLACS1 and EgLACS9 protein probable locates on the outer surface of peroxisomal membrane,and catalyzes long-chain fatty acids into CoA esters,then supplies such acyl-CoA to the FATP transporter.They may also take part in the storage of lipid during fruit development in oil palm.The characterization of EgLACSl will provide a molecular basis for the study of acyl-coenzyme A synthetase-mediated lipid synthesis and degradation in oil palm.