Publications

• The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5alpha. Heterologous expression of Spirulina-desaturase genes.

  Apiradee H., Kalyanee P., Pongsathon P., Patcharaporn D., Matura S., Sanjukta S., Supapon C., Morakot T.

  The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system. In this report ... >> More

  The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system. In this report, the authors describe the expression of the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins. The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively. Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina platensis. The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where the enzyme substrates were provided in appropriate concentration. This indicates that the delta9 and delta12 desaturases were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an E. coli host cell using its cofactors system. In contrast, delta6 desaturase activity can be detected only in vitro where electron carriers are provided. This suggests that while this enzyme is expressed in the heterologous host in its active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the system of Spirulina platensis. << Less

  Mol. Biol. Rep. 31:177-189(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning of delta12- and delta6-desaturases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae.

  Huang Y.-S., Chaudhary S., Thurmond J.M., Bobik E.G. Jr., Yuan L., Chan G.M., Kirchner S.J., Mukerji P., Knutzon D.S.

  Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substr ... >> More

  Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast. << Less

  Lipids 34:649-659(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of delta12-fatty acid desaturase from arachidonic acid-producing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae.

  Sakuradani E., Kobayashi M., Ashikari T., Shimizu S.

  Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fun ... >> More

  Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid. Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean). The evolutionary implications of these enzymes will be discussed. The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S. cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%). Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A. oryzae. The M. alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase. << Less

  Eur. J. Biochem. 261:812-820(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae.

  Covello P.S., Reed D.W.

  The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported. Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomy ... >> More

  The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported. Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomyces cerevisiae cells and were confirmed to be linoleic acid and delta 9, delta 12-hexadecadienoic acid by gas chromatography-mass spectrometry. << Less

  Plant Physiol. 111:223-226(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isolation and functional characterization of two independently-evolved fatty acid Delta12-desaturase genes from insects.

  Zhou X.-R., Horne I., Damcevski K., Haritos V., Green A., Singh S.

  We report the first isolation and characterization of insect fatty acid Delta12-desaturase genes, AdD12Des from house cricket (Acheta domesticus) and TcD12Des from the red flour beetle (Tribolium castaneum), responsible for the production of linoleic acid from oleic acid. Sequence analysis shows t ... >> More

  We report the first isolation and characterization of insect fatty acid Delta12-desaturase genes, AdD12Des from house cricket (Acheta domesticus) and TcD12Des from the red flour beetle (Tribolium castaneum), responsible for the production of linoleic acid from oleic acid. Sequence analysis shows the cricket and flour beetle Delta12-desaturase genes have evolved independently from all previously known Delta12-desaturases and are much more closely related to the archetypal stearoyl-Coenzyme A-acting desaturase from rat than to the phospholipid-acting Delta12-desaturases widely reported in plants. Phylogenetic and functional analysis indicates the cricket AdD12Des gene may have evolved from an ancestral Delta9-desaturase. By contrast, the beetle Delta12-desaturase is distantly related to the cricket genes and beetle Delta9-desaturases suggesting evolution by an independent route. Linoleic acid has key physiological roles in insects and this is the first report of genes capable of producing this essential fatty acid in higher animals. << Less

  Insect Mol. Biol. 17:667-676(2008) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Recent advances in the biochemistry and molecular biology of fatty acyl desaturases.

  Tocher D.R., Leaver M.J., Hodgson P.A.

  Prog Lipid Res 37:73-117(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.

  Dyer J.M., Chapital D.C., Kuan J.C., Mullen R.T., Turner C., McKeon T.A., Pepperman A.B.

  The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional ana ... >> More

  The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity. << Less

  Plant Physiol. 130:2027-2038(2002) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation.

  Wada H., Gombos Z., Murata N.

  The sensitivity (or tolerance) of plants to chilling determines their choice of natural habitat and also limits the worldwide production of crops. Although the molecular mechanism for chilling sensitivity has long been debated, no definitive conclusion has so far been reached about its nature. A p ... >> More

  The sensitivity (or tolerance) of plants to chilling determines their choice of natural habitat and also limits the worldwide production of crops. Although the molecular mechanism for chilling sensitivity has long been debated, no definitive conclusion has so far been reached about its nature. A probable hypothesis, however, is that chilling injury is initiated by phase transition of lipids of cellular membranes, as demonstrated for cyanobacteria, which serve as a model system for the plant cells. Because the phase transition temperature depends on the degree of unsaturation of fatty acids of the membrane lipids, it is predicted that the chilling tolerance of plants can be altered by genetically manipulating fatty-acid desaturation by introducing double bonds into fatty acids of membrane lipids. Here we report the cloning of a gene for the plant-type desaturation (termed desA). The introduction of this gene from a chilling-resistant cyanobacterium, Synechocystis PCC6803, into a chilling-sensitive cyanobacterium, Anacystis nidulans, increases the tolerance of the recipient to low temperature. << Less

  Nature 347:200-203(1990) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Study of the delta 12-desaturase system of Lipomyces starkeyi.

  Lomascolo A., Dubreucq E., Galzy P.

  The specific activity of the microsomal delta 12-desaturase system, which transforms oleic acid into linoleic acid, was about 16 pmol/min/mg protein. However, most of the total activity was nonsedimentable even after a 200000 x g centrifugation for 100 min. The study of various physicochemical par ... >> More

  The specific activity of the microsomal delta 12-desaturase system, which transforms oleic acid into linoleic acid, was about 16 pmol/min/mg protein. However, most of the total activity was nonsedimentable even after a 200000 x g centrifugation for 100 min. The study of various physicochemical parameters showed that this enzymatic complex, functioning optimally between pH 7 and 8, had low thermal stability. Ca2+ which may cause an aggregation of the microsomes, and Hg2+ completely inhibited the activity, whereas Mg2+, Mn2+, and Zn2+ were activators. The delta 12-desaturase system was relatively specific toward oleic acid, though isomers of this fatty acid also had an action, either as substrates or as competitive inhibitors, on the activity of the system. The study of the effect of the exogenous oleoyl-CoA and elaidoyl-CoA on the specific activity of the delta 12-desaturase system showed a preference toward oleoyl-CoA. << Less

  Lipids 31:253-259(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of the delta 12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate.

  Borgeson C.E., de Renobales M., Blomquist G.J.

  The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut t ... >> More

  The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut tissue. In incubations of microsomal preparations from fat body tissues with [1-14C]18:1-CoA, increased amounts of [1-14C]18:2 were found with increasing time and protein concentration. The form of the substrate for the delta 12 desaturase was determined to be 18:1-CoA by comparing activity towards [1-14C]18:1-CoA and [1-14C]18:1 transesterified to phospholipid. Ozonolysis of the 18:2 formed from [1-14C]oleoyl-CoA followed by radio-gas-liquid chromatography gave one labeled peak, 9-oxononanoate, which showed that the product of the delta 12 desaturase is the physiologically important isomer, 18:2(n - 6). << Less

  Biochim Biophys Acta 1047:135-140(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.