| Long chain polyunsaturated fatty acids have many health benefits for human body. In addition to the traditional source of fish oil, LC-PUFAs biosynthesis in oil crops is a prospective alternative source. In the effort of metabolic engineering to produce LC-PUFAs in plants using lipid-linked desaturases, multiple rounds of acyl exchanges of substrate intermediates between PC and acyl-CoA pool are required because desaturases typically use PC as substrate whereas elongases use CoA as substrate, which finally results in low yields of LC-PUFAs.LPCAT which belong to LPLAT is supposed to play an important role in the process of acyl exchange. However, little is known about plant LPCAT and the mechanism of acyl exchange between PC and CoA pool is still not yet clear.In this study, genes encoding LPCATs from Nicotiana benthamiana and Phaeodactylum tricornutum were cloned and characterized. Substrate specificity of LPCATs was determined using yeast microsome as enzyme source. LPCATs and different types of fatty acid desaturases and elongase were co-expressed in yeast. Fatty acids, CoA and lipidomics were analyzed to address how LPCAT affects acyl exchange of substrate intermediates and the amount of LC-PUFAs that accumulate in recombinant yeast. The major results are described as follows:(1) Lysophospholipid acyltransferase genes NbLPCAT1 and NbLPCAT2 encoding proteins belonging to the MBOAT acyltransferase family were cloned from Nicotiana benthamiana. LysoPAF sensitivity assay showed that both NbLPCAT1 and NbLPCAT2 have LPCAT activity. Phospholipid analysis of recombinant yeast showed that NbLPCAT1 and NbLPCAT2 had a preference for 16:0-LPC, 16:1-LPC and 18:1-LPC as acyl acceptor, and18:2-CoA and 18:3-CoA as acyl donor.In vitro enzymatic assay demonstrated that NbLPCAT1 showed a preference for LPC and unsaturated C18-CoAs. NbLPCAT2 showed a high LPAAT activity towards 18:3-CoA and a relatively low LPCAT activity. Expression pattern of NbLPCAT1 and NbLPCAT2 was analyzed using quantitative RT-PCR. The results showed that expression level of NbLPCAT1 and NbLPCAT2 was highest in flowers and the expression of NbLPCAT1 and Nb LPCAT2 in other tissues is ubiquitous.(2) Microalga PtLPCAT gene was cloned and characterized from Phaeodactylum tricornutum that accumulates large amounts of EPA(20:5). NbLPCAT1, NbLPCAT2 and PtLPCAT were co-expressed in yeast with different type of fatty acid desaturases and elongase. These desaturases and elongase include: phospholipid-linked desaturases(PtD6 and PtD5) from Phaeodactylum tricornutum, CoA-dependent desaturases(OtD6 and MsD5) from Ostreococcus tauri and Micromonas pusilla, and elongase(PSE) from Physcomitrella patens.Three different types of recombinant yeast were constructed and functionally analyzed. These recombinant yeast co-express: a) CoA-dependent desaturases and elongase; b) phospholipid-linked desaturases and elongase;(c) phospholipid-linked desaturases, elongase, and LPCAT. The results showed that the end-product of LC-PUFAs in a)-type recombinant yeast is the highest, c)-type recombinant yeast accumulates higher amounts of end-product of LC-PUFAs and intermediates produced by △6-desaturases, when compared with that of b)-type recombinant yeast. This result indicates that LPCAT could improve acyl exchange between PC and CoA pool to an extent, and thus partially alleviate the acyl exchange bottleneck when phospholipid-linked desaturases were used to reconstruct LC-PUFAs synthesis.(3) Lipidomic analysis of of different types of recombinant yeast showed that in c)-type recombinant yeast, the amounts of 18:2 and 18:3 at the position of sn-1, sn-2 of PC were higher compared to that in b)-type recombinant yeast.The result suggested that expression of LPCAT could enhance the amounts of end-product of LC-PUFAs and intermediates produced by △6-desaturation reaction. It further verified substrate specificities of both NbLPCAT1 and NbLPCAT2, and also showed that NbLPCAT1 exhibited no evident preference for acyl position of LPC. |
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