Fatty acid desaturases of cyanobacteria and the modification of membrane lipid unsaturation.

The physical properties of biological membranes depend on the fatty acid unsaturation of membrane lipids.Cyanobacteria respond to a decrease in ambient temperature by desaturating fatty acids of membrane lipids to compensate the decrease in membrane fluidity at low temperature.Fatty acid desaturases are enzymes that introduce double bonds into hydrocarbon chain of fatty acids.The enzymes play an important role to control the fluidity of membrane lipids in the acclimation of cyanobacterial cells to low temperature.<br /> In the cyanobacterium,Synechocystis PCC68037,polyunsaturated fatty acids are synthesized by sequential introduction of double bonds into Cl8fatty acids esterified to the snー1 position of glycerolipids by four distinct desaturases, and each of the desaturases catalyzes the desaturation of fatty acids at a specific position: the △6,△9,△12 or(1)3 position.At the beginning of the present studies, the desA gene that encodes the △12 desaturase had been isolated from Synechocystis PCC6803【Wada,H.,Gombos,Z.and Murata,N.(1990) Nature 347:200-203】,but no other desaturase genes of cyanobacteria,or of higher plants had been reported.<br /> In order to resolve the role of unsaturation of membrane lipids,it is necessary to control the degree of membrane lipid unsaturation by genetic manipulation of the fatty acid desaturases.In addition,the structural information of desaturases is requisite to understand the reaction mechanism of the fatty acid desaturation.<br /> The author isolated cyanobacterial desaturase genes: the △12 desaturase genes(desA) from three strains of cyanobacteria,i.e.,<br /> Synechocystis PCC6714,Synechococcus PCC7002 and Anabaena variabilis,the ω3 desaturase gene(desB) from the cyanobacterium,Synechocystis PCC6803; and the △9 desaturase genes(desC) from A.variabilis and Synechocystis PCC6803.The membrane一lipid unsaturation of cyanobacteria was altered by manipulating the isolated desaturase genes.<br /> Cyanobacterial desA genes were isolated from Synechocystis PCC6714,Synechococcus PCC7002 and A.variabilis by cross-hybridization with DNA probes derived from the desA gene of Synechocystis PCC6803.It appeared that the desA genes of Synechocystis PCC6714,Synechococcus PCC7002 and A.variabilis encode proteins of 349.347 and 350 amino acid residues,respectively.The amino acid sequences of the products of the desA genes revealed the presence of four conserved domains.The transformation of Synechococcus PCC7942 with the desA genes from the three strains was associated with acquisition of the ability to introduce the second double bond at the △12 position of fatty acids.<br /> Cyanobacteria respond to a decease intemperature by desaturating fatty acids of membrane lipids to compensate the decrease in membrane fluidity.Among various desaturation reactions in cyanobacteria,the desaturation at the ω3 position of fatty acids is the most sensitive to the change in ambient temperature.The author isolated a gene for the ω3 desaturase,desB,from the cyanobacterium,Synechocystis PCC6803,using a heterologous hybridization probe derived from the desA gene of the same cyanobacterium.The desB gene encodes a protein of 359 amino-acid residues and of molecular mass of 41.9kDa.In order to manipulate the fatty acid unsaturation of membrane lipids,the desB gene in Synechocystis PCC6803 was mutated by insertion of the Kanamycin-resistance gene cartridge.The resultant mutant was unable to desaturate fatty acids at the ω3 position.On the other hand,the desA and desB genes were introduced into cells of another cyanobacterium,Synechococcus PCC7942,which can desaturate fatty acids of membrane lipids only at the △9 position. The resultant transformant was capable of desaturatin9 fatty acids at the Δ97,△12 and ω3 positions.These results demonstrate that the disruption and the introduction of the desB gene enable us to genetically manipulate the fatty acid unsaturation of membrane lipids in cyanobacteria.<br /> In cyanobacteria,oleic acid is synthesized by introducing a double bond into stearic acid bound to polar glycerolipids.This biosynthetic pathway in cyanobacteria is distinct from the other organisms.丁he author found an open reading frame in the nucleotide sequence of the 5´ upstream region of the desA gene of A.variabilis,and its deduced amino-acid sequence showed similarity to stearoyl-CoA desaturases of rat,mouse and Saccharmyces cerevisiae.Thus,the author postulated that the open reading frame of A.variabilis encodes the cyanobacterial △9 desaturase.The gene was designated desC.Using a DNA fragment derived from the desA gene of A,variabilis,another desC gene was isolated from Synechocystis PCC6803.Upon expression of the desC gene of Synechocystis PCC6803 in Escherichia coli,the E.coli cells accumulated oleic acid,which is not synthesized in the wild‐type cells of E.coli.The deduced amino-acid sequence of the desC gene of Synechocystis PCC6803 shows significant similarity to the stearoyl一CoA desaturases ...

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総研大甲第92号

博士論文

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