Cloning And Functional Analysis Of Genes For Long-Chain Fatty Acid Acyl-CoA Synthetase Protein Family From Phaeodactylum Tricornutum

Phaeodactylum tricornutum is a single-celled oleaginous diatom and it is a model species fordiatom biology. Long chain fatty acid acyl CoA synthetase (LACS) catalyzes the esterification offree long-chain fatty acid to form fatty acyl CoAs, which can been used for triacylglycerol (TAG)biosynthesis, β-oxidation and other biochemical reactions.In this study, genes for LACS protein family (ptacsl) from the diatom P. tricornutum werecloned and biochemically characterized. P. tricornutum LACS protein family contains fivemembers: PtACSL15. Analyses of their functions were performed in the yeast double mutantstrain YB525(faa1Δfaa4Δ). The activity of PtACSL was measured using three fatty acidsubstrates with different chain length and number of double bonds. Gene expression patterns ofptacsl1-5were analyzed under different culture conditions using real-time quantitative RT-PCR.The effect of expression of PtACSL5on the accumulation of docosahexaenoic acid (DHA) inTAG was also evaluated in the yeast faa4Δ mutant. The main results are described as follows:(1) The LACS candidate proteins from P. tricornutum were screened using BLAST.Analysis of amino acid sequences of PtACSLs indicated that all five PtACSLs contain severalconserved domains: AMP binding domain, LACS signature motif, and ACSL-specific linkerdomain. The plasmids harboring each of five ptacsl genes and the empty vector (pYES2/CT) weretransformed into the yeast double mutant YB525(faa1Δfaa4Δ). Their expression was induced byglactose and was verified by Western blot. In the media containing both oleate and cerulenin,growth of YB525strain expressing either PtACSL1or PtACSL4was restored. Confocalmicroscope was used to monitor the intracellular accumulation of the fluorescent long-chain fattyacid analog C1-BODIPY-C12in transgenic yeast strains. The result showed that fluorescencecould be observed within the cells of transgenic yeast strain expressing ptacsl1or ptacsl4,indicating that PtACSL1or PtACSL4could restore fatty acid uptake in the yeast mutant. Nile redwas used to stain live yeast cells and it was found that yeast mutant strain expressing PtACSL1orPtACSL4showed significantly increased intracellular accumulation of storage lipids whencompared to that of control of empty vector. This result indicates that PtACSL1and PtACSL4might play an important role in trafficking intracellular synthesized fatty acids into the pathway ofstorage lipid synthesis. Enzymatic assay showed that each of recombinant PtACSL proteinsexhibited certain activity when oleic acid, linolenic acid and docosahexaenoic acid were used assubstrates.(2) Expression pattern of ptacsl was analyzed using Real-time PCR. Expression level of eachptacsl was significantly decreased within60hours of stationary-phase-exit in P. tricornutum cells.Supplementing oleic acid in the medium could slightly up-regulated the expression of each ofptacsl genes within60hours. Expression level of ptacsl1was four times that of control afternitrogen starvation for two days, indicating that ptacsl1might play a role in increased lipidaccumulation upon nitrogen starvation. (3) It was reported that expression of a long-chain fatty acid acyl-CoA synthetase fromThalassiosira pseudonana (TplascA) in the yeast mutant faa4Δ could enhance DHA content inTAG by six-fold compared to that of control. Ptacsl5, the P. tricornutum homolog of Tplacsa wasalso heterologously expressed in the yeast mutant faa4Δ to evaluate the effect of DHAincorporation into TAG molecule. The result showed the amount of DHA incorporated into TAGin transgenic yeast was10.6times that of the control when DHA was supplemented at a finalconcentration of50μM, indicating that the expression of PtACSL5could significantly increasethe incorporation of DHA into TAG.