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Sample records for acid desaturase sfat-1-transgenic

  1. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  2. Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances

    PubMed Central

    Lee, Je Min; Lee, Hyungjae; Kang, SeokBeom; Park, Woo Jung

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism. PMID:26742061

  3. Fatty Acid Desaturases, Polyunsaturated Fatty Acid Regulation, and Biotechnological Advances.

    PubMed

    Lee, Je Min; Lee, Hyungjae; Kang, SeokBeom; Park, Woo Jung

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) are considered to be critical nutrients to regulate human health and development, and numerous fatty acid desaturases play key roles in synthesizing PUFAs. Given the lack of delta-12 and -15 desaturases and the low levels of conversion to PUFAs, humans must consume some omega-3 and omega-6 fatty acids in their diet. Many studies on fatty acid desaturases as well as PUFAs have shown that fatty acid desaturase genes are closely related to different human physiological conditions. Since the first front-end desaturases from cyanobacteria were cloned, numerous desaturase genes have been identified and animals and plants have been genetically engineered to produce PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Recently, a biotechnological approach has been used to develop clinical treatments for human physiological conditions, including cancers and neurogenetic disorders. Thus, understanding the functions and regulation of PUFAs associated with human health and development by using biotechnology may facilitate the engineering of more advanced PUFA production and provide new insights into the complexity of fatty acid metabolism. PMID:26742061

  4. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  5. A newly discovered member of the fatty acid desaturase gene family: a non-coding, antisense RNA gene to delta5-desaturase.

    PubMed

    Dreesen, Thomas D; Adamson, Aaron W; Tekle, Michael; Tang, Chongren; Cho, Hyekung P; Clarke, Steven D; Gettys, Thomas W

    2006-08-01

    The rate limiting steps in the conversion of 18-carbon unsaturated fatty acids to 20- and 22-carbon products are catalyzed by two desaturase enzymes (Delta5-desaturase and Delta6-desaturase) found within a lipid desaturase gene cluster. Careful examination of this cluster revealed the existence of a conventionally spliced (human) and an intronless (mouse and rat) non-coding RNA gene, reverse Delta5-desaturase, which is transcribed from the opposite strand of the Delta5-desaturase gene. The 654 bp human reverse Delta5-desaturase transcript contains 269 nucleotides that are complementary to exon 1 and intron 1 of the Delta5-desaturase transcript, and the 3'-end of this sequence contains a 143 nucleotide stretch that is 100% complementary to the 5'-end of the Delta5-desaturase. The rat and mouse transcripts are 1355 and 690 bp long and complementary to a portion of the first intron and the entire first exon of their respective Delta5-desaturases. All reverse Delta5-desaturase transcripts contain several stop codons in all frames suggesting that they do not encode a peptide. Reverse Delta5-desaturase RNA was detected in all rat tissues where Delta5-desaturase is expressed, and the transition between fasting and refeeding produced a significant increase in reverse Delta5-desaturase RNA relative to Delta5-desaturase mRNA. Transient expression of reverse Delta5-desaturase in CHO cells stably transformed with Delta5-desaturase produced a modest decrease in Delta5-desaturase mRNA (30%), but lowered Delta5-desaturase enzymatic activity by >70%. More importantly, a diet enriched in fish oil produced a reciprocal increase in reverse Delta5-desaturase mRNA and decrease in Delta5-desaturase mRNA that was accompanied by a 5-6-fold decrease in Delta5-desaturase enzyme activity. These findings support a significant role for reverse Delta5-desaturase as a natural antisense regulator of Delta5-desaturase. PMID:16846730

  6. Light-induced expression of fatty acid desaturase genes

    PubMed Central

    Kis, Mihály; Zsiros, Otto; Farkas, Tibor; Wada, Hajime; Nagy, Ferenc; Gombos, Zoltán

    1998-01-01

    In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases. PMID:9539715

  7. Soybean fatty acid desaturase pathway: reponses to temperature changes and pathogen infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stearoyl-acyl carrier protein-desaturase, omega-6 desaturase, and omega-3 fatty acid desaturase genes are present as multiple copies in the soybean genome as expected given the evidence from cytogenetics, genetic mapping, and genomic sequencing that soybean is a paleopolyploid species that underwent...

  8. A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid.

    PubMed Central

    Pereira, Suzette L; Huang, Yung-Sheng; Bobik, Emil G; Kinney, Anthony J; Stecca, Kevin L; Packer, Jeremy C L; Mukerji, Pradip

    2004-01-01

    Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors. Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA. This activity differs from that of the known omega3-desaturases from any organism. Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops. PMID:14651475

  9. ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use

    PubMed Central

    Wang, Mingxuan; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Wei; Chen, Yong Q.

    2014-01-01

    The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms. PMID:24177732

  10. Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids.

    PubMed

    Liu, Weitian; Jiao, Hongmei; Murray, Nancy C; O'Connor, Marion; Roelofs, Wendell L

    2002-01-22

    Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, that makes E11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene precursor is made available by beta oxidation of E11-16 acid with a subsequent two-carbon loss to E9-14 acid. A functional assay using a baculovirus expression system required addition of myristic acid and E9-14 acid precursors to demonstrate the unusual regiospecificity and stereospecificity of this desaturase. The amino acid sequence of this desaturase has approximately 61% identity to that of Z11-desaturases from two other insect species, and only approximately 48% identity to the metabolic Z9-desaturases in those species. A pheromone-gland Z9-desaturase gene also was found with the light brown apple moth that differed in its deduced amino acid sequence (66% identity) with the metabolic Z9-desaturase from fat body in this species. PMID:11805319

  11. Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

    PubMed

    Saito, T; Ochiai, H

    1999-10-01

    cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds. PMID:10504413

  12. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  13. Mutagenesis of the borage Delta(6) fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Shewry, P; Napier, J

    2000-12-01

    The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase. PMID:11171152

  14. Co-expression of the borage Delta 6 desaturase and the Arabidopsis Delta 15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean.

    PubMed

    Eckert, Helene; La Vallee, Brad; Schweiger, Bruce J; Kinney, Anthony J; Cahoon, Edgar B; Clemente, Tom

    2006-10-01

    Two relatively rare fatty acids, gamma-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, alpha-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Delta(6) desaturase and an Arabidopsis Delta(15) desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Delta(6) desaturase event with the Delta(15) desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean beta-conglycinin promoter. Soybean events that carried only the Delta(15 )desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids. PMID:16718484

  15. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

    PubMed

    Thambugala, Dinushika; Cloutier, Sylvie

    2014-11-01

    Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax. PMID:24871199

  16. Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Castel, A; Shewry, P R; Napier, J A

    2001-07-01

    Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue. PMID:11457919

  17. Functional expression of a Δ12 fatty acid desaturase gene from spinach in transgenic pigs

    PubMed Central

    Saeki, Kazuhiro; Matsumoto, Kazuya; Kinoshita, Mikio; Suzuki, Iwane; Tasaka, Yasushi; Kano, Koichiro; Taguchi, Yoshitomo; Mikami, Koji; Hirabayashi, Masumi; Kashiwazaki, Naomi; Hosoi, Yoshihiko; Murata, Norio; Iritani, Akira

    2004-01-01

    Linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Δ12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Δ12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were ≈10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained ≈20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases. PMID:15067141

  18. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    NASA Astrophysics Data System (ADS)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2015-11-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  19. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    NASA Astrophysics Data System (ADS)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2016-07-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  20. Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls.

    PubMed

    Klinkenberg, Joern; Faist, Hanna; Saupe, Stefanie; Lambertz, Sophie; Krischke, Markus; Stingl, Nadja; Fekete, Agnes; Mueller, Martin J; Feussner, Ivo; Hedrich, Rainer; Deeken, Rosalia

    2014-02-01

    Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ9-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions. PMID:24368335

  1. Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

    PubMed

    Rutter, Andrew J; Thomas, Katie L; Herbert, Derek; Henderson, R James; Lloyd, David; Harwood, John L

    2002-11-15

    Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Delta(10)-nonadecenoate showed that the enzyme is an n -6 desaturase rather than a Delta(12)-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b (5) as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b (5)) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n -6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n -6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms. PMID:12153399

  2. Functional and safety evaluation of transgenic pork rich in omega-3 fatty acids.

    PubMed

    Tang, Maoxue; Qian, Lili; Jiang, Shengwang; Zhang, Jian; Song, Pengkun; Chen, Yaoxing; Cui, Wentao; Li, Kui

    2014-08-01

    Genetically modified animals rich in omega-3 unsaturated fatty acid offer a new strategy to improve the human health, but at the same time present a challenge in terms of food safety assessment. In this study, we evaluated the function and safety of sFat-1 transgenic pork rich in omega-3 fatty acids in mice by feeding basic diet and diets that contain wild type pork and sFat-1 transgenic pork. Blood biochemistry, haematology, peripheral T cell distributions, bacterial counts, gross necropsy, histopathology and organ weights were performed in mice fed with different doses of wild type and transgenic pork. Results indicated that both low and high dose of wild type and transgenic pork had no significant effect on blood biochemistry, T cell distribution, immunoglobulins and bacterial counts in intestine and feces. However, it was noted that both low and high dose of transgenic pork improved the liver immune system in mice, which is probably due to the beneficial contribution of high level of the "good" fatty acids in transgenic pork. There is no significant effect of transgenic pork on all other organs in mice. In summary, our study clearly demonstrated that feeding transgenic pork rich in omega-3 fatty acids did not cause any harm to mice, and in fact, improved the liver immune system. PMID:24700396

  3. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco

    SciTech Connect

    Cahoon, E.B.; Shanklin, J.; Ohlrogge, J.B. )

    1992-12-01

    Little is known about the metabolic origin of petroselinic acid (18:1[Delta][sup 6cis]), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the [Delta][sup 9]-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and [Delta][sup 4]-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase. 27 refs., 5 figs.

  4. Fatty acid desaturase gene variants, cardiovascular risk factors, and myocardial infarction in the costa rica study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variation in fatty acid desaturases (FADS) has previously been linked to long-chain polyunsaturated fatty acids (PUFAs) in adipose tissue and cardiovascular risk. The goal of our study was to test associations between six common FADS polymorphisms (rs174556, rs3834458, rs174570, rs2524299, r...

  5. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.

    PubMed Central

    Cahoon, E B; Shanklin, J; Ohlrogge, J B

    1992-01-01

    Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase. Images PMID:1454797

  6. Changes in fatty acid composition in plant tissues expressing a mammalian delta9 desaturase.

    PubMed

    Moon, H; Hazebroek, J; Hildebrand, D F

    2000-05-01

    Plant tissues expressing a mammalian stearoyl-CoA delta9 desaturase were reported to accumulate delta9 hexadecenoic acid (16:1), normally very minor in most plant tissues. The transgenic plants were thoroughly analyzed for alterations of individual lipids in different subcellular sites. Western blot analysis indicated that the animal desaturase was targeted to the microsomes. The delta9 16:1 was incorporated into both the sn-1 and sn-2 positions of all the major membrane lipids tested, indicating that the endoplasmic reticulum acyltransferases do not exclude unsaturated C16 fatty acids from the sn-2 position. In addition to increases in monounsaturated and decreases in saturated fatty acids, accumulation of 16:1 was accompanied by a reduction in 18:3 in all the lipids tested except phosphatidylglycerol, and increases in 18:2 in phospholipids. Total C16 fatty acid content in the galactolipids of the transgenics was significantly higher than that in the control, but those in the phospholipids were unchanged. In transgenics, delta11 18:1 was detected in the sn-1 position of the lipids tested except phosphatidylinositol and phosphatidylserine. Introduction of the animal desaturase, controlled by a seed-specific phaseolin promoter, into soybean somatic embryo resulted in a significant reduction in saturated fatty acids. Such effects were greater in cotyledons than hypocotyl-radicles. This study demonstrated that the animal desaturase can be used to decrease the levels of saturated fatty acids in a crop plant. PMID:10907781

  7. INHIBITION OF FATTY ACID DESATURASES IN Drosophila melanogaster LARVAE BLOCKS FEEDING AND DEVELOPMENTAL PROGRESSION.

    PubMed

    Wang, Yiwen; da Cruz, Tina Correia; Pulfemuller, Alicia; Grégoire, Stéphane; Ferveur, Jean-François; Moussian, Bernard

    2016-05-01

    Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program. PMID:27037621

  8. Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems.

    PubMed

    Bai, Shuangyi; Wallis, James G; Denolf, Peter; Browse, John

    2016-07-01

    Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon-optimized DSG open reading frame with an endoplasmic-reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently-detected single amino acid change, Q240R, yielded a nearly 25-fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60-fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine-rich regions known to be critical for electron transfer mediated by these di-iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522-1530. © 2016 Wiley Periodicals, Inc. PMID:26724425

  9. Inventory of Fatty Acid Desaturases in the Pennate Diatom Phaeodactylum tricornutum

    PubMed Central

    Dolch, Lina-Juana; Maréchal, Eric

    2015-01-01

    The diatom Phaeodactylum is rich in very long chain polyunsaturated fatty acids (PUFAs). Fatty acid (FA) synthesis, elongation, and desaturation have been studied in depth in plants including Arabidopsis, but for secondary endosymbionts the full picture remains unclear. FAs are synthesized up to a chain length of 18 carbons inside chloroplasts, where they can be incorporated into glycerolipids. They are also exported to the ER for phospho- and betaine lipid syntheses. Elongation of FAs up to 22 carbons occurs in the ER. PUFAs can be reimported into plastids to serve as precursors for glycerolipids. In both organelles, FA desaturases are present, introducing double bonds between carbon atoms and giving rise to a variety of molecular species. In addition to the four desaturases characterized in Phaeodactylum (FAD2, FAD6, PtD5, PtD6), we identified eight putative desaturase genes. Combining subcellular localization predictions and comparisons with desaturases from other organisms like Arabidopsis, we propose a scheme at the whole cell level, including features that are likely specific to secondary endosymbionts. PMID:25786062

  10. Structural organization of fatty acid desaturase loci in linseed lines with contrasting linolenic acid contents.

    PubMed

    Thambugala, Dinushika; Ragupathy, Raja; Cloutier, Sylvie

    2016-07-01

    Flax (Linum usitatissimum L.), the richest crop source of omega-3 fatty acids (FAs), is a diploid plant with an estimated genome size of ~370 Mb and is well suited for studying genomic organization of agronomically important traits. In this study, 12 bacterial artificial chromosome clones harbouring the six FA desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b from the conventional variety CDC Bethune and the high linolenic acid line M5791 were sequenced, analysed and compared to determine the structural organization of these loci and to gain insights into the genetic mechanisms underlying FA composition in flax. With one gene every 3.2-4.6 kb, the desaturase loci have a higher gene density than the genome's average of one gene per 7.8-8.2 kb. The gene order and orientation across the two genotypes were generally conserved with the exception of the sad1 locus that was predicted to have additional genes in CDC Bethune. High sequence conservation in both genic and intergenic regions of the sad and fad2b loci contrasted with the significant level of variation of the fad2a and fad3 loci, with SNPs being the most frequently observed mutation type. The fad2a locus had 297 SNPs and 36 indels over ~95 kb contrasting with the fad2b locus that had a mere seven SNPs and four indels in ~110 kb. Annotation of the gene-rich loci revealed other genes of known role in lipid or carbohydrate metabolic/catabolic pathways. The organization of the fad2b locus was particularly complex with seven copies of the fad2b gene in both genotypes. The presence of Gypsy, Copia, MITE, Mutator, hAT and other novel repeat elements at the desaturase loci was similar to that of the whole genome. This structural genomic analysis provided some insights into the genomic organization and composition of the main desaturase loci of linseed and of their complex evolution through both tandem and whole genome duplications. PMID:27142663

  11. Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

    PubMed

    Libisch, B; Michaelson, L V; Lewis, M J; Shewry, P R; Napier, J A

    2000-12-29

    The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets. PMID:11162428

  12. Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

    PubMed

    Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2013-10-15

    Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. PMID:23796520

  13. Cloning and transcriptional analysis of Crepis alpina fatty acid desaturases affecting the biosynthesis of crepenynic acid.

    PubMed

    Nam, Jeong-Won; Kappock, T Joseph

    2007-01-01

    Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Delta12 position of linoleic acid, forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR), acetylenase transcripts were identified in non-seed C. alpina tissues, which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds, genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5), or that compete for substrate (FAD3), were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels, and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 10(4)-fold higher acetylenase expression in developing seeds than in flower heads. Collectively, the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Delta12 acetylenase in a polyacetylene biosynthetic pathway, so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product, crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes. PMID:17329262

  14. Applications of Stereospecifically-labeled Fatty Acids in Oxygenase and Desaturase Biochemistry

    PubMed Central

    Brash, Alan R.; Schneider, Claus; Hamberg, Mats

    2012-01-01

    Oxygenation and desaturation reactions are inherently associated with the abstraction of a hydrogen from the fatty acid substrate. Since the first published application in 1965, stereospecific placement of a labeled hydrogen isotope (deuterium or tritium) at the reacting carbons has proven a highly effective strategy for investigating the chemical mechanisms catalyzed by lipoxygenases, hemoprotein fatty acid dioxygenases including cyclooxygenases, cytochromes P450, and also the desaturases and isomerases. This review presents a synopsis of all published studies through 2010 on the synthesis and use of stereospecifically labeled fatty acids (70 references), and highlights some of the mechanistic insights gained by application of stereospecifically labeled fatty acids. PMID:21971646

  15. Associations between a fatty acid desaturase gene polymorphism and blood arachidonic acid compositions in Japanese elderly.

    PubMed

    Horiguchi, Sayaka; Nakayama, Kazuhiro; Iwamoto, Sadahiko; Ishijima, Akiko; Minezaki, Takayuki; Baba, Mamiko; Kontai, Yoshiko; Horikawa, Chika; Kawashima, Hiroshi; Shibata, Hiroshi; Kagawa, Yasuo; Kawabata, Terue

    2016-02-01

    We investigated whether the single nucleotide polymorphism rs174547 (T/C) of the fatty acid desaturase-1 gene, FADS1, is associated with changes in erythrocyte membrane and plasma phospholipid (PL) long-chain polyunsaturated fatty acid (LCPUFA) composition in elderly Japanese participants (n=124; 65 years or older; self-feeding and oral intake). The rs174547 C-allele carriers had significantly lower arachidonic acid (ARA; n-6 PUFA) and higher linoleic acid (LA, n-6 PUFA precursor) levels in erythrocyte membrane and plasma PL (15% and 6% ARA reduction, respectively, per C-allele), suggesting a low LA to ARA conversion rate in erythrocyte membrane and plasma PL of C-allele carriers. α-linolenic acid (n-3 PUFA precursor) levels were higher in the plasma PL of C-allele carriers, whereas levels of the n-3 LCPUFAs eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were unchanged in erythrocyte membrane and plasma PL. Thus, rs174547 genotypes were significantly associated with different ARA compositions of the blood of elderly Japanese. PMID:26869086

  16. Polyunsaturated fatty acid supplementation reverses cystic fibrosis-related fatty acid abnormalities in CFTR-/- mice by suppressing fatty acid desaturases.

    PubMed

    Njoroge, Sarah W; Laposata, Michael; Boyd, Kelli L; Seegmiller, Adam C

    2015-01-01

    Cystic fibrosis patients and model systems exhibit consistent abnormalities in metabolism of polyunsaturated fatty acids that appear to play a role in disease pathophysiology. Recent in vitro studies have suggested that these changes are due to overexpression of fatty acid desaturases that can be reversed by supplementation with the long-chain polyunsaturated fatty acids docosahexaenoate and eicosapentaenoate. However, these findings have not been tested in vivo. The current study aimed to test these results in an in vivo model system, the CFTR(-/-) knockout mouse. When compared with wild-type mice, the knockout mice exhibited fatty acid abnormalities similar to those seen in cystic fibrosis patients and other model systems. The abnormalities were confined to lung, ileum and pancreas, tissues that are affected by the disease. Similar to in vitro models, these fatty acid changes correlated with increased expression of Δ5- and Δ6-desaturases and elongase 5. Dietary supplementation with high-dose free docosahexaenoate or a combination of lower-dose docosahexaenoate and eicosapentaenoate in triglyceride form corrected the fatty acid abnormalities and reduced expression of the desaturase and elongase genes in the ileum and liver of knockout mice. Only the high-dose docosahexaenoate reduced histologic evidence of disease, reducing mucus accumulation in ileal sections. These results provide in vivo support for the hypothesis that fatty acid abnormalities in cystic fibrosis result from abnormal expression and activity of metabolic enzymes in affected cell types. They further demonstrate that these changes can be reversed by dietary n-3 fatty acid supplementation, highlighting the potential therapeutic benefit for cystic fibrosis patients. PMID:25448610

  17. Linoleic acid biosynthesis and characterization of the. Delta. sup 12 desaturase in insects

    SciTech Connect

    Cripps, C.

    1988-01-01

    De novo biosynthesis of linoleic acid was demonstrated in vivo in 8 of 32 insect species examined, including both holometabolous and hemimetabolous species. The incorporation of (1-{sup 14}C) acetate into linoleic acid was demonstrated by radio-gas-liquid chromatography (radio-GLC), and in selected species by radio-high-performance liquid chromatography, silver nitrate thin-layer chromatography, radio-GLC and GLC linked to mass spectrometry of ozonolysis products. Analysis of the ozonolysis products clearly demonstrated that the entire molecule was labeled and that synthesis of linoleate was de novo from acetate. The in vivo incorporation of (1-{sup 14}C)acetate into lipid was monitored during the final three stadia of both male and female house crickets, Acheta domesticus. Characterization of the {Delta}{sup 12}-desaturase showed that, in the house cricket, this enzyme is microsomal and requires a reduced pyridine dinucleotide as a cofactor, with NADPH the preferred electron donor. The optimal substrate concentration for desaturation is about 40 uM. Addition of the microsomal supernatant, MgCl{sub 2} or ATP did not enhance activity. The form of the substrate for the desaturase, oleic acid, was determined and appears to be a CoA derivative, as is true for most animal desaturases, rather than a complex lipid, as it is in plants.

  18. [Cloning and expression of a delta6-fatty acid desaturase gene from Rhizopus stolonifer in Saccharomyces cervisiae].

    PubMed

    Lu, He; Chai, You-rong; Zhang, Xue-kun; Lei, Tian-gang; Li, Jia-na

    2007-02-01

    Fatty acid composition of fungi is analysed through the gas chromatography( GC) technique. With specific activity a novel enzyme delta6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R. stolonifer was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1475bp full-length cDNA, designated as RnD6D here, with high homology to fungal delta6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid(GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid. PMID:17436625

  19. IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF THE MOSS PHYSCOMITRELLA PATENS DELTA5-DESATURASE GENE INVOLVED IN ARACHIDONIC AND EICOSAPENTAENOIC ACID BIOSYNTHESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. In general, these C20 polyunsaturated fatty acids are synthesized from linoleic and alpha-linolenic acids, respectively, by a series of reactions catalyzed by a delta6-desaturase, an ...

  20. Contribution of the different omega-3 fatty acid desaturase genes to the cold response in soybean

    PubMed Central

    Andreu, Vanesa; Alfonso, Miguel

    2012-01-01

    This study analysed the contribution of each omega-3 desaturase to the cold response in soybean. Exposure to cold temperatures (5 °C) did not result in great modifications of the linolenic acid content in leaf membrane lipids. However, an increase in the GmFAD3A transcripts was observed both in plant leaves and soybean cells whereas no changes in GmFAD3B or GmFAD3C expression levels were detected. This increase was reversible and accompanied by the accumulation of an mRNA encoding a truncated form of GmFAD3A (GmFAD3A-T), which originated from alternative splicing of GmFAD3A in response to cold. When the expression of plastidial omega-3 desaturases was analysed, a transient accumulation of GmFAD7-2 mRNA was detected upon cold exposure in mature soybean trifoliate leaves while GmFAD7-1 transcripts remained unchanged. No modification of the GmFAD8-1 and GmFAD8-2 transcripts was observed. The functionality of GmFAD3A, GmFAD3B, GmFAD3C and GmFAD3A-T was examined by heterologous expression in yeast. No activity was detected with GmFAD3A-T, consistent with the absence of one of the His boxes necessary for desaturase activity. The linolenic acid content of Sacharomyces cerevisiae cells overexpressing GmFAD3A or GmFAD3B was higher when the cultures were incubated at cooler temperatures, suggesting that reticular desaturases of the GmFAD3 family, and more specifically GmFAD3A, may play a role in the cold response, even in leaves. The data point to a regulatory mechanism of omega-3 fatty acid desaturases in soybean affecting specific isoforms in both the plastid and the endoplasmic reticulum to maintain appropriate levels of linolenic acid under low temperature conditions. PMID:22865909

  1. New layers in understanding and predicting α-linolenic acid content in plants using amino acid characteristics of omega-3 fatty acid desaturase.

    PubMed

    Zinati, Zahra; Zamansani, Fatemeh; Hossein KayvanJoo, Amir; Ebrahimi, Mahdi; Ebrahimi, Mansour; Ebrahimie, Esmaeil; Mohammadi Dehcheshmeh, Manijeh

    2014-11-01

    α-linolenic acid (ALA) is the most frequent omega-3 in plants. The content of ALA is highly variable, ranging from 0 to 1% in rice and corn to >50% in perilla and flax. ALA production is strongly correlated with the enzymatic activity of omega-3 fatty acid desaturase. To unravel the underlying mechanisms of omega-3 diversity, 895 protein features of omega-3 fatty acid desaturase were compared between plants with high and low omega-3. Attribute weighting showed that this enzyme in plants with high omega-3 content has higher amounts of Lys, Lys-Phe, and Pro-Asn but lower Aliphatic index, Gly-His, and Pro-Leu. The Random Forest model with Accuracy criterion when run on the dataset pre-filtered with Info Gain algorithm was the best model in distinguishing high omega-3 content based on the frequency of Lys-Lys in the structure of fatty acid desaturase. Interestingly, the discriminant function algorithm could predict the level of omega-3 only based on the six important selected attributes (out of 895 protein attributes) of fatty acid desaturase with 75% accuracy. We developed "Plant omega3 predictor" to predict the content of α-linolenic acid based on structural features of omega-3 fatty acid desaturase. The software calculates the 6 key structural protein features from imported Fasta sequence of omega-3 fatty acid desaturase or utilizes the imported features and predicts the ALA content using discriminant function formula. This work unravels an underpinning mechanism of omega-3 diversity via discovery of the key protein attributes in the structure of omega-3 desaturase offering a new approach to obtain higher omega-3 content. PMID:25199845

  2. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii.

    PubMed

    Chi, Xiaoyuan; Zhang, Xiaowen; Guan, Xiangyu; Ding, Ling; Li, Youxun; Wang, Mingqing; Lin, Hanzhi; Qin, Song

    2008-04-01

    Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae). PMID:18545969

  3. Δ12-Fatty acid desaturase from Candida parapsilosis is a multifunctional desaturase producing a range of polyunsaturated and hydroxylated fatty acids.

    PubMed

    Buček, Aleš; Matoušková, Petra; Sychrová, Hana; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

    2014-01-01

    Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina). PMID:24681902

  4. The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

    PubMed Central

    Pédrono, Frédérique; Blanchard, Hélène; Kloareg, Maela; D'andréa, Sabine; Daval, Stéphanie; Rioux, Vincent; Legrand, Philippe

    2010-01-01

    In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3. PMID:19752397

  5. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

    1998-01-06

    Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  6. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  7. A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids.

    PubMed

    Sayanova, Olga; Haslam, Richard; Guschina, Irina; Lloyd, David; Christie, William W; Harwood, John L; Napier, Johnathan A

    2006-12-01

    The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist. PMID:16950768

  8. Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3

    PubMed Central

    Garba, Lawal; Mohamad Ali, Mohd Shukuri; Oslan, Siti Nurbaya; Rahman, Raja Noor Zaliha Raja Abd

    2016-01-01

    Fatty acid desaturase enzymes play an essential role in the synthesis of unsaturated fatty acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- fatty acid desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-fatty acid desaturase capable of increasing the total amount of cellular unsaturated fatty acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-fatty acid desaturase-like protein which was actively expressed in E. coli. PMID:27494717

  9. Elevated delta-6 desaturase (FADS2) expression in the postmortem prefrontal cortex of schizophrenic patients: relationship with fatty acid composition.

    PubMed

    Liu, Yanhong; Jandacek, Ronald; Rider, Therese; Tso, Patrick; McNamara, Robert K

    2009-04-01

    Although emerging evidence suggests that schizophrenia (SZ) is associated with peripheral and central polyunsaturated fatty acid (PUFA) deficits, there is currently nothing known about the expression of genes that mediate PUFA biosynthesis in SZ patients. Here we determined Delta5 desaturase (FADS1), Delta6 desaturase (FADS2), elongase (HELO1 [ELOVL5]), peroxisomal (PEX19), and Delta9 desaturase (stearoyl-CoA desaturase, SCD) mRNA expression, and relevant fatty acid product:precursor ratios as estimates of enzyme activities, in the postmortem prefrontal cortex (PFC) of patients with SZ (n=20) and non-psychiatric controls (n=20). After correction for multiple comparisons, FADS2 mRNA expression was significantly greater in SZ patients relative to controls (+36%, p=0.002), and there was a positive trend found for FADS1 (+26%, p=0.15). No differences were found for HELO1 (+10%, p=0.44), PEX19 (+12%, p=0.44), or SCD (-6%, p=0.85). Both male (+34%, p=0.02) and female (+42%, p=0.02) SZ patients exhibited greater FADS2 mRNA expression relative to same-gender controls. Drug-free SZ patients (+37%, p=0.02), and SZ patients treated with typical (+40%, p=0.002) or atypical (+31%, p=0.04) antipsychotics, exhibited greater FADS2 mRNA expression relative to controls. Consistent with increased Delta6 desaturase activity, SZ patients exhibited a greater 20:3/18:2 ratio (+20%, p=0.03) and a positive trend was found for 20:4/18:2 (+13%, p=0.07). These data demonstrate abnormal, potentially compensatory, elevations in Delta6 desaturase (FADS2) expression in the PFC of SZ patients that are independent of gender and antipsychotic medications. Greater Delta6 desaturase expression and activity could have implications for central prostaglandin synthesis and proinflammatory signaling. PMID:19195843

  10. [Desaturases of fatty acids (FADS) and their physiological and clinical implication].

    PubMed

    Žák, Aleš; Slabý, Adolf; Tvrzická, Eva; Jáchymová, Marie; Macášek, Jaroslav; Vecka, Marek; Zeman, Miroslav; Staňková, Barbora

    2016-01-01

    States associated with insulin resistance, as overweight/obesity, type 2 diabetes mellitus (DM2), cardiovascular diseases (CVD), some cancers and neuropsychiatric diseases are characterized with a decrease of long-chain polyunsaturated fatty acids (LC-PUFA) levels. Amounts of LC-PUFA depend on the exogenous intake of their precursors [linoleic (LA) and α-linolenic acid (ALA)] and by rate of their metabolism, which is influenced by activities of enzymes, such as Δ6-desaturase (D6D, FADS2), D5D, FADS1, elongases (Elovl2, -5, 6).Altered activities of D5D/D6D were described in plenty of diseases, e.g. neuropsychiatric (depressive disorders, bipolar disorder, dementia), metabolic (obesity, metabolic syndrome, DM2) and cardiovascular diseases (arterial hypertension, coronary heart disease), inflammatory states and allergy (Crohns disease, atopic eczema) or some malignancies. Similar results were obtained in studies dealing with the associations between genotypes/haplotypes of FADS1/FADS2 and above mentioned diseases, or interactions of dietary intake of LA and ALA on one hand and of the polymorphisms of minor allels of FADS1/FADS2, usually characterized by lower activities, on the other hand.The decrease of the desaturases activities leads to decreased concentrations of products with concomitant increased concentrations of substrates. Associations of some SNP FADS with coronary heart disease, concentrations of plasma lipids, oxidative stress, glucose homeostasis, and inflammatory reaction, were described. Experimental studies on animal models and occurrence of rare diseases, associated with missing or with marked fall activities of D5D/D6D emphasized the significance of desaturases for healthy development of organism as well as for pathogenesis of some disease. PMID:27088787

  11. Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator's Sex Pheromone.

    PubMed

    Sedeek, Khalid E M; Whittle, Edward; Guthörl, Daniela; Grossniklaus, Ueli; Shanklin, John; Schlüter, Philipp M

    2016-06-01

    Mimicry illustrates the power of selection to produce phenotypic convergence in biology [1]. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species [2-4]. This involves mimicry of visual, tactile, and chemical signals of females [2-7], especially their sex pheromones [8-11]. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11-13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O. exaltata and closely related species, such as O. sphegodes [13-16]. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5's reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. This change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution. PMID:27212404

  12. Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus.

    PubMed

    Sakuradani, Eiji; Shimizu, Sakayu

    2003-04-01

    We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid. For one of the two delta 6-desaturases, designated as delta 6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two delta 6-desaturases, designated as delta 6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella delta 6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast delta 9-desaturase, which has the corresponding domain at the C-terminus. The full-length delta 6II cDNA was expressed in A. oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II RNA on 2, 3, and 4 days in M. alpina 1S-4, respectively. M. alpina 1S-4 is the first fungus to be confirmed to have two functional delta 6-desaturase genes. PMID:12784608

  13. Indices of fatty acid desaturase activity in healthy human subjects: effects of different types of dietary fat.

    PubMed

    Vessby, Bengt; Gustafsson, Inga-Britt; Tengblad, Siv; Berglund, Lars

    2013-09-14

    Δ9-Desaturase (stearoyl-CoA desaturase 1, SCD-1) regulates the desaturation of SFA, mainly stearic and palmitic, to MUFA. Δ6-Desaturase (D6D) and Δ5-desaturase (D5D) are involved in the metabolism of linoleic and α-linolenic acid to polyunsaturated metabolites. The objective of the present study was to study the effects of different types of dietary fat on indices of fatty acid desaturase (FADS) activity (evaluated as product:precursor ratios) in plasma and skeletal muscle in human subjects. A high SCD-1 index has been related to obesity and metabolic disorders, while the D5D index is associated with insulin sensitivity. Fatty acid composition of serum and skeletal muscle lipids was analysed by GLC during a randomised, controlled, 3-month dietary intervention in healthy subjects. A comparison of the effects of a diet containing butter fat (SFA, n 17) with a diet containing monounsaturated fat (MUFA, n 17), keeping all other dietary components constant, showed a reduced SCD-1 activity index by 20% on the MUFA diet compared with the SFA diet assessed in serum cholesteryl esters. The D6D and D5D indices remained unaffected. Supplementation with long-chain n-3 fatty acids reduced the SCD-1 index by a similar magnitude while the D6D index decreased and the D5D index increased. It is concluded that changes in the type of fat in the diet affect the indices of FADS activity in serum and skeletal muscle in human subjects. The desaturase activity indices estimated from the serum lipid ester composition are significantly related to corresponding indices studied in skeletal muscle phospholipids. PMID:23414551

  14. Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds.

    PubMed

    Dehghan Nayeri, Fatemeh; Yarizade, Kazem

    2014-08-01

    Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds. PMID:24816719

  15. Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

    PubMed Central

    Buček, Aleš; Matoušková, Petra; Vogel, Heiko; Šebesta, Petr; Jahn, Ullrich; Weißflog, Jerrit; Svatoš, Aleš; Pichová, Iva

    2015-01-01

    For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds. PMID:26417103

  16. Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase.

    PubMed

    Buček, Aleš; Matoušková, Petra; Vogel, Heiko; Šebesta, Petr; Jahn, Ullrich; Weißflog, Jerrit; Svatoš, Aleš; Pichová, Iva

    2015-10-13

    For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds. PMID:26417103

  17. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    PubMed

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity. PMID:24880119

  18. Glycine max cultivar Dare chloroplast fatty acid desaturase 7 (FAD7) gene, complete cds; nuclear gene for chloroplast product

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomic sequence of soybean GmFAD7 (2455 nucleotides) was determined and reported to GenBank and assigned the accession number HM769340. The structure and deduced amino acid sequence of soybean FAD7 is similar to other higher plant plastidal omega-3 desaturases: 8 exons and 7 introns, predicted...

  19. Glycine max cultivar Dare chloroplast fatty acid desaturase 8 (FAD8) gene, complete cds; nuclear gene for chloroplast product

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomic sequence of soybean GmFAD8 (2480 nucleotides) was determined and reported to GenBank and assigned the accession number HM769341. The structure and deduced amino acid sequence of soybean FAD8 is similar to other higher plant plastidal omega-3 desaturases: 8 exons and 7 introns, predicted...

  20. Soybean plastidal omega-3 fatty acid desaturase genes GmFAD7 and GmFAD8: structure and expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomic structure and deduced protein sequence of soybean FAD7 and FAD8 have features similar to higher plant plastidal '-3 desaturases: 8 exons and 7 introns, predicted proteins of 453 amino acid residues containing three conserved histidine motifs, amino terminal chloroplast transit peptides, ...

  1. Effect of temperature on microsomal omega-3 linoleate desaturase gene expression and linolenic acid content in developing soybean seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post-translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the tr...

  2. Delta 5 fatty acid desaturase upregulates the synthesis of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum.

    PubMed

    Peng, Kun-Tao; Zheng, Cun-Ni; Xue, Jiao; Chen, Xiao-Yan; Yang, Wei-Dong; Liu, Jie-Sheng; Bai, Weibin; Li, Hong-Ye

    2014-09-01

    Microalgae are important primary producers in the marine ecosystem and excellent sources of lipids and other bioactive compounds. The marine diatom Phaeodactylum tricornutum accumulates eicosapentaenoic acid (EPA, 20:5n-3) as its major component of fatty acids. To improve the EPA production, delta 5 desaturase, which plays a role in EPA biosynthetic pathway, was characterized in P. tricornutum. An annotated delta 5 desaturase PtD5b gene was cloned and overexpressed in P. tricornutum. The transgene was integrated into the genome demonstrated by Southern blot, and the overexpression of PtD5b was verified by qPCR and Western blot analysis. Fatty acid composition exhibited a significant increase in the unsaturated fatty acids. Monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) showed an increase of 75% and 64%, respectively. In particular, EPA showed an increase of 58% in engineered microalgae. Meanwhile, neutral lipid content showed an increase up to 65% in engineered microalgae. More importantly, engineered cells showed a similar growth rate with the wild type, thus keeping high biomass productivity. This work provides an effective way to improve the production of microalgal value-added bioproducts by metabolic engineering. PMID:25109502

  3. [Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco].

    PubMed

    Li, Ming-Chun; Liu, Li; Hu, Guo-Wu; Xing, Lai-Jun

    2003-03-01

    Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were

  4. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    PubMed Central

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  5. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

    PubMed

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  6. New Insight into Phaeodactylum tricornutum Fatty Acid Metabolism. Cloning and Functional Characterization of Plastidial and Microsomal Δ12-Fatty Acid Desaturases1[w

    PubMed Central

    Domergue, Frédéric; Spiekermann, Patricia; Lerchl, Jens; Beckmann, Christoph; Kilian, Oliver; Kroth, Peter G.; Boland, Wilhem; Zähringer, Ulrich; Heinz, Ernst

    2003-01-01

    In contrast to 16:3 plants like rapeseed (Brassica napus), which contain α-linolenic acid (18:3Δ9,12,15) and hexadecatrienoic acid (16:3Δ7,10,13) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and a different isomer of hexadecatrienoic acid (16:3Δ6,9,12). In this report, we describe the characterization of two cDNAs having sequence homology to Δ12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Δ12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1Δ9), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the ω9-fatty acids 18:1Δ9 and 16:1Δ7, in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1Δ9. Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3Δ6,9,12) is of plastidial origin. PMID:12692324

  7. A More Desirable Balanced Polyunsaturated Fatty Acid Composition Achieved by Heterologous Expression of Δ15/Δ4 Desaturases in Mammalian Cells

    PubMed Central

    Zhu, Guiming; Ou, Qin; Zhang, Tao; Jiang, Xudong; Sun, Guozhi; Zhang, Ning; Wang, Kunfu; Fang, Heng; Wang, Mingfu; Sun, Jie; Ge, Tangdong

    2013-01-01

    Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway–a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition – a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health. PMID:24391980

  8. Identification and heterologous expression of a Δ4-fatty acid desaturase gene from Isochrysis sphaerica.

    PubMed

    Guo, Bing; Jiang, Mulan; Wan, Xia; Gong, Yangmin; Liang, Zhuo; Hu, Chuanjiong

    2013-10-28

    The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops. PMID:23851273

  9. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

    PubMed

    Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2014-09-01

    Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement. PMID:24851712

  10. Fatty Acid Desaturase Mutants of Yeast: Growth Requirements and Electron Spin Resonance Spin-Label Distribution

    PubMed Central

    Wisnieski, Bernadine J.; Kiyomoto, Richard K.

    1972-01-01

    Two respiratory-sufficient and one respiratory-deficient (nuclear petite) strains of yeast Δ9-desaturase mutants were analyzed to determine which fatty acids would serve as replacements for the naturally occurring fatty acids, 16:1 Δ9cis and 18:1 Δ9cis. The requirement can be satisfied by several fatty acids differing in double-bond position, steric configuration, chain length, and degree of unsaturation. The features common to growth-supporting fatty acids are presented and the effects of varying the carbon source and temperature are considered. In addition, we illustrate several pitfalls encountered in membrane studies which exploit lipid-requiring organisms. Since the membrane fatty acid composition of these mutants can be modified readily, electron spin resonance spectroscopy is used to compare membranes of mutant strains enriched for different fatty acids. The lipid distribution pattern of the most commonly employed electron spin resonance spin-label, 12-nitroxide stearate, was ascertained and compared to that of 18:1 Δ9cis. PMID:4333377

  11. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    PubMed

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  12. Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice

    PubMed Central

    Roqueta-Rivera, Manuel; Stroud, Chad K.; Haschek, Wanda M.; Akare, Sandeep J.; Segre, Mariangela; Brush, Richard S.; Agbaga, Martin-Paul; Anderson, Robert E.; Hess, Rex A.; Nakamura, Manabu T.

    2010-01-01

    Delta-6 desaturase-null mice (−/−) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The −/− males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type (+/+) and −/− males at weaning until 16 weeks of age (n = 3–5). A breeding success rate of DHA-supplemented −/− was comparable to +/+. DHA-fed −/− showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed −/−, but DPAn6 remained depleted. In AA-fed −/−, AA was restored at the +/+ level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of +/+ and DHA-fed −/−, whereas it diminished in −/− and AA-fed −/−, suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA. PMID:19690334

  13. Obesity resistance and deregulation of lipogenesis in Δ6-fatty acid desaturase (FADS2) deficiency

    PubMed Central

    Stoffel, Wilhelm; Hammels, Ina; Jenke, Britta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Odenthal, Margarete; Thevis, Mario

    2014-01-01

    Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2−/− mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2−/− mutant, the molecular basis of which is unknown. Phospholipidomic profiling of membrane systems of fads2−/−mice revealed diacylglycerol-structures, deprived of PUFAs but substituted with surrogate eicosa-5,11,14-trienoic acid. ω6-Arachidonic (AA) and ω3-docosahexaenoic acid (DHA) supplemented diets transformed fads2−/− into AA-fads2−/− and DHA-fads2−/− mutants. Severely altered phospholipid-bilayer structures of subcellular membranes of fads2−/− liver specifically interfered with maturation of transcription factor sterol-regulatory-element-binding protein, the key regulator of lipogenesis and lipid homeostasis. This study strengthens the concept that specific PUFA-substituted membrane phospholipid species are critical constituents of the structural platform operative in lipid homeostasis in normal and disease conditions. PMID:24378641

  14. Transgenic expression of delta-6 and delta-15 fatty acid desaturases enhances omega-3 polyunsaturated fatty acid accumulation in Synechocystis sp. PCC6803

    PubMed Central

    2014-01-01

    Background Polyunsaturated fatty acids (PUFAs), which contain two or more double bonds in their backbone, are the focus of intensive global research, because of their nutritional value, medicinal applications, and potential use as biofuel. However, the ability to produce these economically important compounds is limited, because it is both expensive and technically challenging to separate omega-3 polyunsaturated fatty acids (ω-3 PUFAs) from natural oils. Although the biosynthetic pathways of some plant and microalgal ω-3 PUFAs have been deciphered, current understanding of the correlation between fatty acid desaturase content and fatty acid synthesis in Synechocystis sp. PCC6803 is incomplete. Results We constructed a series of homologous vectors for the endogenous and exogenous expression of Δ6 and Δ15 fatty acid desaturases under the control of the photosynthesis psbA2 promoter in transgenic Synechocystis sp. PCC6803. We generated six homologous recombinants, harboring various fatty acid desaturase genes from Synechocystis sp. PCC6803, Gibberella fujikuroi and Mortierella alpina. These lines produced up to 8.9 mg/l of α-linolenic acid (ALA) and 4.1 mg/l of stearidonic acid (SDA), which are more than six times the corresponding wild-type levels, at 20°C and 30°C. Thus, transgenic expression of Δ6 and Δ15 fatty acid desaturases enhances the accumulation of specific ω-3 PUFAs in Synechocystis sp. PCC6803. Conclusions In the blue-green alga Synechocystis sp. PCC6803, overexpression of endogenous and exogenous genes encoding PUFA desaturases markedly increased accumulation of ALA and SDA and decreased accumulation of linoleic acid and γ-linolenic acid. This study lays the foundation for increasing the fatty acid content of cyanobacteria and, ultimately, for producing nutritional and medicinal products with high levels of essential ω-3 PUFAs. PMID:24581179

  15. Dietary arachidonic acid and docosahexaenoic acid regulate liver fatty acid desaturase (FADS) alternative transcript expression in suckling piglets.

    PubMed

    Wijendran, Vasuki; Downs, Ian; Srigley, Cynthia Tyburczy; Kothapalli, Kumar S D; Park, Woo Jung; Blank, Bryant S; Zimmer, J Paul; Butt, C M; Salem, Norman; Brenna, J Thomas

    2013-10-01

    Molecular regulation of fatty acid desaturase (Fads) gene expression by dietary arachidonic acid (ARA) and docosahexaenoic acid (DHA) during early post-natal period, when the demand for long chain polyunsaturated fatty acids (LC-PUFA) is very high, has not been well defined. The objective of the current study was to determine regulation of liver Fads1, Fads2 and Fads3 classical (CS) and alternative transcripts (AT) expression by dietary ARA and DHA, within the physiological range present in human breast milk, in suckling piglets. Piglets were fed one of six milk replacer formula diets (formula-reared groups, FR) with varying ARA and DHA content from days 3-28 of age. The ARA/DHA levels of the six formula diets were as follows (% total fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. The control maternal-reared (MR) group remained with the dam. Fads1 expression was not significantly different between FR and MR groups. Fads2 expression was down-regulated significantly in diets with 1:1 ratio of ARA:DHA, compared to MR. Fads2 AT1 expression was highly correlated to Fads2 expression. Fads3 AT7 was the only Fads3 transcript sensitive to dietary LC-PUFA intake and was up-regulated in the formula diets with lowest ARA and DHA contents compared to MR. Thus, the present study provides evidence that the proportion of dietary ARA:DHA is a significant determinant of Fads2 expression and LC-PUFA metabolism during the early postnatal period. Further, the data suggest that Fads3 AT7 may have functional significance when dietary supply of ARA and DHA are low during early development. PMID:24075244

  16. Genomic structural analysis of porcine fatty acid desaturase cluster on chromosome 2.

    PubMed

    Taniguchi, Masaaki; Arakawa, Aisaku; Motoyama, Michiyo; Nakajima, Ikuyo; Nii, Masahiro; Mikawa, Satoshi

    2015-04-01

    Fatty acid composition is an economically important trait in meat-producing livestock. To gain insight into the molecular genetics of fatty acid desaturase (FADS) genes in pigs, we investigated the genomic structure of the porcine FADS gene family on chromosome 2. We also examined the tissue distribution of FADS gene expression. The genomic structure of FADS family in mammals consists of three isoforms FADS1, FADS2 and FADS3. However, porcine FADS cluster in the latest pig genome assembly (Sscrofa 10.2) containing some gaps is distinct from that in other mammals. We therefore sought to determine the genomic structure, including the FADS cluster in a 200-kbp range by sequencing gap regions. The structure we obtained was similar to that in other mammals. We then investigated the porcine FADS1 transcription start site and identified a novel isoform named FADS1b. Phylogenetic analysis revealed that the three members of the FADS cluster were orthologous among mammals, whereas the various FADS1 isoforms identified in pigs, mice and cattle might be attributable to species-specific transcriptional regulation with alternative promoters. Porcine FADS1b and FADS3 isoforms were predominantly expressed in the inner layer of the subcutaneous adipose tissue. Additional analyses will reveal the effects of these functionally unknown isoforms on fatty acid composition in pig fat tissues. PMID:25409917

  17. Screening and molecular identification of overproducing γ-linolenic acid fungi and cloning the delta 6-desaturase gene.

    PubMed

    Lu, He; Zhu, Yu

    2015-01-01

    This research aims at isolating and identifying γ-linolenic acid (GLA)-producing fungi in the traditional Chinese salt-fermented soybean food, Douchi, from Yongchuan, People's Republic of China. In this study, Rhizopus oryzae DR3 was identified as a novel fungal species that produces large amounts of GLA. A full-length cDNA, designated as RoD6 D, with high homology to fungal △6 fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1,176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6 D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N-terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6 D exhibited △6 fatty acid desaturase activity that led to the accumulation of GLA. The results show that Douchi contains a large natural diverse composition, and some strains could be selected as starters for functional fermented foods. This study has also laid a foundation for developing functional Douchi products for further research. PMID:25169017

  18. Delta-6 desaturase from borage converts linoleic acid to gamma-linolenic acid in HEK293 cells.

    PubMed

    Chen, Qing; Nimal, Jonathan; Li, Wanli; Liu, Xia; Cao, Wenguang

    2011-07-01

    Gamma-linolenic acid (GLA, 18:3 n6) is an essential polyunsaturated fatty acid of the omega-6 family and is found to be effective in prevention and/or treatment of various health problems. In this study, we evaluated the possibility of increasing γ-linolenic acid contents in mammalian cells using the delta-6 gene from Borago officinalis. The borage Δ6-desaturase gene (sDelta-6) was codon-optimized and introduced into HEK293 cells by lipofectin transfection. Co-expression of GFP with sDelta-6 and RT-PCR analysis indicated that sDelta-6 could be expressed in mammalian cells. Subsequently, the heterologous expression of borage Δ6-desaturase was evaluated by fatty acid analysis. Total cellular lipid analysis of transformed cells fed with linoleic acid (LA 18:2 n6) as a substrate showed that the expression of sDelta-6 resulted in an 228-483% (p<0.05) increase of GLA when compared with that in the control cells. The highest conversion efficiency of LA into GLA in sDelta-6(+) cells was 6.9 times higher than that in the control group (11.59% vs. 1.69%; p<0.05). Our present work demonstrated that the sDelta-6 gene from borage could be functionally expressed in mammalian cells, and could convert LA into GLA. Furthermore, this study may pave the way to generate transgenic livestock that can synthesise GLA. PMID:21679695

  19. The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants.

    PubMed

    Sayanova, Olga; Ruiz-Lopez, Noemi; Haslam, Richard P; Napier, Johnathan A

    2012-02-01

    The role of acyl-CoA-dependent Δ6-desaturation in the heterologous synthesis of omega-3 long-chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl-CoA Δ6-desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6-desaturated acyl-CoAs, in contrast to the phospholipid-dependent Δ6-desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl-CoA Δ6-desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid-dependent Δ6-desaturase. The use of acyl-CoA-dependent Δ6-desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ-linolenic acid in total seed lipids. Expression of acyl-CoA Δ6-desaturases resulted in increased distribution of long-chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6-desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6-desaturated fatty acids. This study provides evidence for the efficacy of using acyl-CoA-dependent Δ6-desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega-3 LC-PUFAs. PMID:21902798

  20. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

  1. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

    1998-01-06

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

  2. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells

    PubMed Central

    Park, Hui Gyu; Park, Woo Jung; Kothapalli, Kumar S. D.; Brenna, J. Thomas

    2015-01-01

    Docosahexaenoic acid (DHA) is a Δ4-desaturated C22 fatty acid and the limiting highly unsaturated fatty acid (HUFA) in neural tissue. The biosynthesis of Δ4-desaturated docosanoid fatty acids 22:6n-3 and 22:5n-6 are believed to proceed via a circuitous biochemical pathway requiring repeated use of a fatty acid desaturase 2 (FADS2) protein to perform Δ6 desaturation on C24 fatty acids in the endoplasmic reticulum followed by 1 round of β-oxidation in the peroxisomes. We demonstrate here that the FADS2 gene product can directly Δ4-desaturate 22:5n-3→22:6n-3 (DHA) and 22:4n-6→22:5n-6. Human MCF-7 cells lacking functional FADS2-mediated Δ6-desaturase were stably transformed with FADS2, FADS1, or empty vector. When incubated with 22:5n-3 or 22:4n-6, FADS2 stable cells produce 22:6n-3 or 22:5n-6, respectively. Similarly, FADS2 stable cells when incubated with d5-18:3n-3 show synthesis of d5-22:6n-3 with no labeling of 24:5n-3 or 24:6n-3 at 24 h. Further, both C24 fatty acids are shown to be products of the respective C22 fatty acids via elongation. Our results demonstrate that the FADS2 classical transcript mediates direct Δ4 desaturation to yield 22:6n-3 and 22:5n-6 in human cells, as has been widely shown previously for desaturation by fish and many other organisms.—Park, H. G., Park, W. J., Kothapalli, K. S. D., Brenna, J. T. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells. PMID:26065859

  3. Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

    PubMed

    Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

    2016-01-01

    Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. PMID:26514651

  4. Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato.

    PubMed

    Yu, Chao; Wang, Hua-Sen; Yang, Sha; Tang, Xian-Feng; Duan, Ming; Meng, Qing-Wei

    2009-01-01

    An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 degrees C), but inhibited by high temperature (40 degrees C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F(v)/F(m)) and the O(2) evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress. PMID:19648018

  5. Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid {delta}5 desaturase gene.

    PubMed

    Kobayashi, Takumi; Sakaguchi, Keishi; Matsuda, Takanori; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2011-06-01

    Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C(16:0)), n - 6 docosapentaenoic acid (DPA) (C(22:5)(n) (- 6)), and docosahexaenoic acid (DHA) (C(22:6)(n) (- 3)), with eicosapentaenoic acid (EPA) (C(20:5)(n) (- 3)) and arachidonic acid (AA) (C(20:4)(n) (- 6)) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C(20:4)(n) (- 3)) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C(20:3)(n) (- 6)) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs. PMID:21478316

  6. Increase of Eicosapentaenoic Acid in Thraustochytrids through Thraustochytrid Ubiquitin Promoter-Driven Expression of a Fatty Acid Δ5 Desaturase Gene▿†

    PubMed Central

    Kobayashi, Takumi; Sakaguchi, Keishi; Matsuda, Takanori; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2011-01-01

    Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C16:0), n − 6 docosapentaenoic acid (DPA) (C22:5n − 6), and docosahexaenoic acid (DHA) (C22:6n − 3), with eicosapentaenoic acid (EPA) (C20:5n − 3) and arachidonic acid (AA) (C20:4n − 6) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C20:4n − 3) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C20:3n − 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs. PMID:21478316

  7. Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

    PubMed

    Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S

    2013-11-01

    Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family. PMID:24062307

  8. Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids*

    PubMed Central

    Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S.

    2013-01-01

    Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family. PMID:24062307

  9. Fatty Acid Desaturase 1 (FADS1) Gene Polymorphisms Control Human Hepatic Lipid Composition

    PubMed Central

    Wang, Libo; Athinarayanan, Shaminie; Jiang, Guanglong; Chalasani, Naga; Zhang, Min; Liu, Wanqing

    2014-01-01

    Fatty Acid Desaturase (FADS) genes and their variants have been associated with multiple metabolic phenotypes including liver enzymes and hepatic fat accumulation but the detailed mechanism remains unclear. We aimed to delineate the role of FADSs in modulating lipid composition in human liver. We performed a targeted lipidomic analysis of a variety of phospholipids, sphingolipids and ceramides among 154 human liver tissue samples. The associations between previously Genome-wide Association Studies (GWAS)-identified six FADS single nucleotide polymorphisms (SNPs) and these lipid levels as well as total hepatic fat content (HFC) were tested. The potential function of these SNPs in regulating transcription of 3 FADS genes (FADS1, FADS2 and FADS3) in the locus was also investigated. We found that while these SNPs were in high linkage disequilibrium (r2 >0.8), the rare alleles of these SNPs were consistently and significantly associated with the accumulation of multiple very-long-chain fatty acids (VLCFAs), with C47H85O13P (C36:4), a phosphatidylinositol (PI) and C43H80O8PN (C38:3), a phosphatidylethanolamine (PE) reached the Bonferroni corrected significance (p<3×10−4). Meanwhile, these SNPs were significantly associated with increased ratios between the more saturated and relatively less saturated forms of VLCFAs, especially between PEs, PIs and phosphatidylcholines (PCs) (p≤3.5×10−6). These alleles were also associated with increased total HFC (p<0.05). Further analyses revealed that these alleles were associated with decreased hepatic expression of FADS1 (p=0.0018 for rs174556), but not FADS2 or FADS3 (p>0.05). Conclusion Our findings revealed critical insight into the mechanism underlying FADS1 and its polymorphisms in modulating hepatic lipid deposition by altering gene transcription and controlling lipid composition in human livers. PMID:25123259

  10. Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids.

    PubMed

    Matsuda, Takanori; Sakaguchi, Keishi; Kobayashi, Takumi; Abe, Eriko; Kurano, Norihide; Sato, Akira; Okita, Yuji; Sugimoto, Shinichi; Hama, Yoichiro; Hayashi, Masahiro; Okino, Nozomu; Ito, Makoto

    2011-10-01

    We isolated a putative desaturase gene from a marine alga, Pinguiochrysis pyriformis MBIC 10872, which is capable of accumulating eicosapentaenoic acid (C20:5(Δ5,8,11,14,17)). The gene possessed an open reading frame of 1,314 bp encoding a putative 437 amino acid residues showing high sequence identity (37-48%) with fungal and nematode Δ12-fatty acid desaturases. Yeast cells transformed with the gene converted endogenous oleic acid (C18:1(Δ9)) to linoleic acid (C18:2(Δ9,12)). However, no double bonds were introduced into other endogenous fatty acids or exogenously added fatty acids. Flag-tagged enzyme was recovered in the micosome fraction when expressed in yeast cells. To express the gene in thraustochytrids, a construct driven by the thraustochytrid-derived ubiquitin promoter was used. Interestingly, exogenously added oleic acid was converted to linoleic acid in the gene transformants but not mock transformants of Aurantiochytrium limacinum mh0186. These results clearly indicate that the gene encodes a microsomal Δ12-fatty acid desaturase and was expressed functionally in not only yeasts but also thraustochytrids. This is the first report describing the heterozygous expression of a fatty acid desaturase in thraustochytrids, and could facilitate a genetic approach towards fatty acid synthesis in thraustochytrids which are expected to be an alternative source of polyunsaturated fatty acids. PMID:21705343

  11. Reducing saturated fatty acids in Arabidopsis seeds by expression of a Caenorhabditis elegans 16:0-specific desaturase.

    PubMed

    Fahy, Deirdre; Scheer, Barbara; Wallis, James G; Browse, John

    2013-05-01

    Plant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT-5, a membrane-bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed-specific promoter. The FAT-5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1∆9; expression of fat-5 reduced the 16:0 content of the seed by two-thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn-1 position. Seeds from a plant line homozygous for FAT-5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value. PMID:23279079

  12. Molecular mapping of genes encoding microsomal omega-6 desaturase enzymes and their cosegregation with QTL affecting oleic acid content in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The microsomal omega-6 desaturase enzymes, which catalyze the desaturation of oleic acid to linoleic acid during fatty acid biosynthesis, are encoded by the FAD2-1 and FAD2-2 genes in soybeans. Breeders aim to incorporate the high oleate trait into soybean germplasm in order to improve the nutrition...

  13. Molecular cloning and ontogenic mRNA expression of fatty acid desaturase in the carnivorous striped snakehead fish (Channa striata).

    PubMed

    Jaya-Ram, Annette; Ishak, Sairatul Dahlianis; Enyu, Yee-Ling; Kuah, Meng-Kiat; Wong, Kah-Loon; Shu-Chien, Alexander Chong

    2011-04-01

    There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues. PMID:21130179

  14. Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage delta6-desaturase.

    PubMed

    Cook, David; Grierson, Don; Jones, Craigh; Wallace, Andrew; West, Gill; Tucker, Greg

    2002-06-01

    The improvement of nutritional quality is one potential application for the genetic modification of plants. One possible target for such manipulation is the modification of fatty acid metabolism. In this work, expression of a borage delta6-desaturase cDNA in tomato (Lycopersicon esculentum L.) has been shown to produce gamma-linolenic acid (GLA; 18:83 delta6,9,12) and octadecatetraenoic acid (OTA; 18:4 delta6,9,12,15) in transgenic leaf and fruit tissue. This genetic modification has also, unexpectedly, resulted in a reduction in the percentage of linoleic acid (LA 18:2 delta9,12) and a concomitant increase in the percentage of alpha-linolenic acid (ALA; 18:3 delta9,12,15) in fruit tissue. These changes in fatty acid composition are thought to be beneficial for human health. PMID:12059112

  15. Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns.

    PubMed

    Dong, Chun-Juan; Cao, Ning; Zhang, Zhi-Gang; Shang, Qing-Mao

    2016-01-01

    Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber. PMID:26938877

  16. Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns

    PubMed Central

    Dong, Chun-Juan; Cao, Ning; Zhang, Zhi-Gang; Shang, Qing-Mao

    2016-01-01

    Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber. PMID:26938877

  17. Differential transcriptional activity of SAD, FAD2 and FAD3 desaturase genes in developing seeds of linseed contributes to varietal variation in α-linolenic acid content.

    PubMed

    Rajwade, Ashwini V; Kadoo, Narendra Y; Borikar, Sanjay P; Harsulkar, Abhay M; Ghorpade, Prakash B; Gupta, Vidya S

    2014-02-01

    Linseed or flax (Linum usitatissimum L.) varieties differ markedly in their seed α-linolenic acid (ALA) levels. Fatty acid desaturases play a key role in accumulating ALA in seed. We performed fatty acid (FA) profiling of various seed developmental stages of ten Indian linseed varieties including one mutant variety. Depending on their ALA contents, these varieties were grouped under high ALA and low ALA groups. Transcript profiling of six microsomal desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B), which act sequentially in the fatty acid desaturation pathway, was performed using real-time PCR. We observed gene specific as well as temporal expression pattern for all the desaturases and their differential expression profiles corresponded well with the variation in FA accumulation in the two groups. Our study points to efficient conversion of intermediate FAs [stearic (SA), oleic (OA) and linoleic acids (LA)] to the final product, ALA, due to efficient action of all the desaturases in high ALA group. While in the low ALA group, even though the initial conversion up to OA was efficient, later conversions up to ALA seemed to be inefficient, leading to higher accumulation of OA and LA instead of ALA. We sequenced the six desaturase genes from the ten varieties and observed that variation in the amino acid (AA) sequences of desaturases was not responsible for differential ALA accumulation, except in the mutant variety TL23 with very low (<2%) ALA content. In TL23, a point mutation in the FAD3A gene resulted into a premature stop codon generating a truncated protein with 291 AA. PMID:24380374

  18. Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

    DOE PAGESBeta

    Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; Inmon, Jay J.; Millhollon, Jon C.; Liechty, Zach; Page, Justin T.; Jenks, Matthew A.; Chapman, Kent D.; Udall, Joshua A.; et al

    2014-11-18

    The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior

  19. Maternal fatty acid desaturase genotype correlates with infant immune responses at 6 months.

    PubMed

    Muc, Magdalena; Kreiner-Møller, Eskil; Larsen, Jeppe M; Birch, Sune; Brix, Susanne; Bisgaard, Hans; Lauritzen, Lotte

    2015-09-28

    Breast milk long-chain PUFA (LCPUFA) have been associated with changes in early life immune responses and may modulate T-cell function in infancy. We studied the effect of maternal fatty acid desaturase (FADS) genotype and breast milk LCPUFA levels on infants' blood T-cell profiles and ex vivo-produced cytokines after anti-CD3/CD28 stimulation of peripheral blood mononuclear cells in 6-month-old infants from the Copenhagen Prospective Study of Asthma in Childhood birth cohort. LCPUFA concentrations of breast milk were assessed at 4 weeks of age, and FADS SNP were determined in both mothers and infants (n 109). In general, breast milk arachidonic acid (AA) levels were inversely correlated with the production of IL-10 (r -0.25; P=0.004), IL-17 (r -0.24; P=0.005), IL-5 (r -0.21; P=0.014) and IL-13 (r -0.17; P=0.047), whereas EPA was positively correlated with the counts of blood regulatory T-cells and cytotoxic T-cells and decreased T-helper cell counts. The minor FADS alleles were associated with lower breast milk AA and EPA, and infants of mothers carrying the minor allele of FADS SNP rs174556 had higher production of IL-10 (r -0.23; P=0.018), IL-17 (r -0.25; P=0.009) and IL-5 (r -0.21; P=0.038) from ex vivo-activated immune cells. We observed no association between T-cell distribution and maternal or infant FADS gene variants. We conclude that increased maternal LCPUFA synthesis and breast milk AA are associated with decreased levels of IL-5, IL-13 (type-2 related), IL-17 (type-17 related) and IL-10 (regulatory immune responses), but not with interferon-γ and TNF-α, which could be due to an effect of the maternal FADS variants on the offspring immune response transferred via breast milk LCPUFA. PMID:26283408

  20. The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)...

  1. TEMPERATURE-SENSITIVE, POST-TRANSLATIONAL REGULATION OF PLANT OMEGA-3 FATTY ACID DESATURASES IS MEDIATED BY THE ER-ASSOCIATED DEGRADATION PATHWAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty acid desaturases (Fad3s) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not upregulated during this adaptive response. Here, we expressed two closely related ...

  2. Arabidopsis Fatty Acid Desaturase FAD2 Is Required for Salt Tolerance during Seed Germination and Early Seedling Growth

    PubMed Central

    Sun, Jian; Li, Bei; Zhu, Qiang; Chen, Shaoliang; Zhang, Hongxia

    2012-01-01

    Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis. PMID:22279586

  3. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes. PMID:27370696

  4. Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress.

    PubMed

    An, Meiling; Mou, Shanli; Zhang, Xiaowen; Zheng, Zhou; Ye, Naihao; Wang, Dongsheng; Zhang, Wei; Miao, Jinlai

    2013-12-01

    The Antarctic ice microalgae Chlamydomonas sp. ICE-L which is highly resistant to salt stress holds promise in providing an alternative species for the production of microalgal oil. We studied the effects of the alga in confrontation with NaCl stress on the growth, oil yield and expression of fatty acid desaturase genes. The growth rate of Chlamydomonas sp. ICE-L decreased with the gradual increase in NaCl concentration. Interestingly, we found that the highest lipid content was achieved at 16‰ NaCl, reaching 23% (w/w). Meanwhile, the expression of Δ9ACPCiFAD increased rapidly while Δ12CiFAD, ω3CiFAD2 and Δ6CiFAD showed a delayed elevation in response to altered salt stress. C18:3 was the dominant PUFA, which account for about 75% TFA in Chlamydomonas sp. ICE-L. Under 96‰ and 128‰ NaCl stress, the content of C20:5 almost approached that of C18:3. In contrast, low salinity enhanced the dominance of C18:3 at the expense of C20:3 and C20:5. PMID:24084208

  5. Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

    SciTech Connect

    Zhang, P.; Shanklin, J.; Burton, J. W.; Upchurch, R. G.; Whittle, E.; Dewey, R. E.

    2008-11-01

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoform of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.

  6. Association of serum fatty acid and estimated desaturase activity with hypertension in middle-aged and elderly Chinese population

    PubMed Central

    Yang, Bo; Ding, Fang; Wang, Feng-Lei; Yan, Jing; Ye, Xiong-Wei; Yu, Wei; Li, Duo

    2016-01-01

    We aimed to investigate the cross-sectional associations of serum fatty acid (FA) and related Δ-desaturase with hypertension among 2,447 community-dwellers aged 35–79 years living in Zhejiang Province, China. Individual FA was determined in serum, Δ5-desaturase (D5D) and Δ6-desaturase (D6D) activities were indirectly estimated by FA product/precursor ratios. Participants in the highest quartile of D5D component scores (20:4n–6, 20:5n–3, 22:6n–3 and D5D) have significantly lower odds of hypertension compared with individuals in the lowest (multivariate-adjusted odds ratio (OR) = 0.68, 95% CI: 0.46–0.98). When further stratified by gender, high D5D component scores were significantly associated with lower odds of hypertension in women (OR = 0.53, 95% CI: 0.35–0.80), but not in men (OR = 0.78, 95% CI: 0.52-1.18). Multivariate-adjusted prevalent OR for an interquartile increment of individual FA and estimated desaturase was 1.27 (95% CI: 1.08–1.50) for 16:0, 1.15 (95% CI: 1.01–1.30) for 16:1n–7, 0.89 (95% CI: 0.80–0.99) for 22:6n–3, 1.32 (95% CI: 1.01–1.72) for D6D (18:3n–6/18:2n–6), and 0.74 (95% CI: 0.56, 0.98) for D5D (20:4n–6/20:3n–6). Present findings suggested that high serum 22:6n–3 and D5D as well as low 16:0, 16:1n–7 and D6D were associated with a low prevalence of hypertension in this Chinese population. PMID:27006169

  7. Expression of Delta(12) fatty acid desaturase during the induced accumulation of the antifungal diene in avocado fruits.

    PubMed

    Wang, Xuejun; Beno-Moualem, Delila; Kobiler, Ilana; Leikin-Frenkel, Alicia; Lichter, Amnon; Prusky, Dov

    2004-11-01

    SUMMARY The preformed (Z,Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (AFD) is the most active antifungal compound in avocado; it affects the quiescence of Colletotrichum gloeosporioides in unripe fruit. One of the genes encoding Delta(12) fatty acid desaturase (avfad12) was hypothesized to take part in the biosynthesis of AFD, and its expression pattern and enzymatic activity were determined in relation to the content of AFD. Using avfad12-3 as a probe, high levels of expression were detected in young fruits and leaves, where the level of AFD was highest. In contrast, Northern analysis of RNA from mature leaves and fruits showed no transcripts from the avfad12 gene family and lower AFD content. The transcripts from the avfad12 gene family, the enzymatic activity of Delta(12) fatty acid desaturase, and the level of AFD in unripe-resistant fruits increased transiently when the fruits were inoculated with C. gloeosporioides or exposed to ethylene (40 microL/L), low temperature (4 degrees C) or 1 mm H(2)O(2), but ripe fruits were not affected. The effect of H(2)O(2) on the transcripts from the avfad12 gene family is of specific importance, because reactive oxygen species were produced by unripe-resistant host fruit soon after inoculation of C. gloeosporioides. In addition, the fungus itself produced H(2)O(2) in culture medium at pH 5.0, which is similar to the pH of unripe-resistant fruit, but not at pH 7.0. Treatments that enhanced Delta(12) fatty acid desaturase activity increased the concentration of the AFD precursor, linoleic acid, and its incorporation into AFD; these treatments also caused a delay in decay development. The present results demonstrate temporal relationships among the transcripts from the avfad12 gene family, the synthesis of the precursor of AFD (linoleic acid), the AFD content and quiescence of C. gloeosporioides in unripe fruits. PMID:20565631

  8. Cloning and functional characterization of Δ6 fatty acid desaturase (FADS2) in Eurasian perch (Perca fluviatilis).

    PubMed

    Geay, F; Tinti, E; Mellery, J; Michaux, C; Larondelle, Y; Perpète, E; Kestemont, P

    2016-01-01

    The Eurasian perch (Perca fluviatilis) is a freshwater carnivorous species of high interest to diversify inland aquaculture. However, little is known about its ability to bioconvert polyunsaturated fatty acids (PUFAs) from plant oils into long chain polyunsaturated fatty acids (LC-PUFAs). In this study, special attention has been given to the fatty acid desaturase 2 (FADS2) which is commonly described to be a rate-limiting enzyme of the LC-PUFA biosynthesis. This work reports on the cloning, tissue expression and functional characterization of the Eurasian perch fads2, but also on the cloning of two alternative splicing transcripts named fads2-AS1 and fads2-AS2. The fads2 cDNA cloned is composed of an open reading frame (ORF) of 1338 nucleotides (nt) and encodes a protein of 445 amino acids. This deduced amino acid sequence displays the typical structure of microsomal FADS2 including two transmembrane domains and an N-terminal cytochrome b5 domain with the "HPGG" motif. Quantitative real-time PCR assay of fads2, fads2-AS1 and fads2-AS2 expressions revealed that the fads2 transcript was mainly expressed in the liver and intestine and exhibited a typical gene expression pattern of freshwater species while fads2-AS1 and fads2-AS2 genes were highly expressed in the brain, followed by the liver and intestine. Functional characterization of Eurasian perch FADS2 in transgenic yeast showed a fully functional Δ6 desaturation activity toward C18 PUFA substrates, without residual Δ5 and Δ8 desaturase activities. PMID:26478265

  9. Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

    PubMed Central

    Jaureguiberry, María S.; Tricerri, M. Alejandra; Sanchez, Susana A.; Finarelli, Gabriela S.; Montanaro, Mauro A.; Prieto, Eduardo D.; Rimoldi, Omar J.

    2014-01-01

    Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis. PMID:24473084

  10. Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii

    PubMed Central

    Lim, Jong-Min; Vikramathithan, Jayaraman; Hwangbo, Kwon; Ahn, Joon-Woo; Park, Youn-Il; Choi, Dong-Woog; Jeong, Won-Joong

    2015-01-01

    Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7. PMID:25699037

  11. Cross-sectional associations of food consumption with plasma fatty acid composition and estimated desaturase activities in Finnish children.

    PubMed

    Venäläinen, Taisa; Schwab, Ursula; Ågren, Jyrki; de Mello, Vanessa; Lindi, Virpi; Eloranta, Aino-Maija; Kiiskinen, Sanna; Laaksonen, David; Lakka, Timo A

    2014-05-01

    Plasma fatty acid (FA) composition is known to be an indicator of dietary fat quality, but the associations of other dietary factors with plasma FA composition remain unknown in children. We investigated the cross-sectional associations of food consumption with the proportions of FA and estimated desaturase activities in plasma cholesteryl esters (CE) and phospholipids (PL) among children. The subjects were a population sample of 423 children aged 6–8 years examined at baseline of The Physical Activity and Nutrition in Children (PANIC) Study. We assessed food consumption by food records and plasma FA composition by gas chromatography. We used linear regression models adjusted for age, sex, physical activity and total energy intake to analyze the associations. A higher consumption of vegetable oil-based margarine (fat 60–80 %) was associated with a higher proportion of linoleic and α-linolenic acids in plasma CE and PL. A higher consumption of high-fiber grain products was related to a lower proportion of oleic acid in CE and PL. The consumption of candy was directly associated with the proportion of palmitoleic and oleic acid in plasma CE. The consumption of vegetable oil-based margarine was inversely associated with estimated stearoyl-CoA-desaturase activity in plasma CE and PL and the consumption of candy was directly related to it in plasma CE. The results of our study suggest that plasma FA composition is not only a biomarker for dietary fat quality but also reflects the consumption of high-fiber grain products and foods high in sugar among children. PMID:24659110

  12. Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

    PubMed

    Zhao, Na; Zhang, Yuan; Li, Qiuqi; Li, Rufang; Xia, Xinli; Qin, Xiaowei; Guo, Huihong

    2015-02-01

    Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants. PMID:25528221

  13. The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

    PubMed Central

    De Smet, Cedric H.; Vittone, Elisa; Scherer, Max; Houweling, Martin; Liebisch, Gerhard; Brouwers, Jos F.; de Kroon, Anton I.P.M.

    2012-01-01

    The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta­king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings. PMID:22323296

  14. Targeted mutation of Δ12 and Δ15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil.

    PubMed

    Bielecka, Monika; Kaminski, Filip; Adams, Ian; Poulson, Helen; Sloan, Raymond; Li, Yi; Larson, Tony R; Winzer, Thilo; Graham, Ian A

    2014-06-01

    We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven Δ12 desaturases and one of three Δ15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Δx+3 activity, while CSFAD3A activity was exclusively at the Δ15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major Δ12 and Δ15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1(Δ9) ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2(Δ6,9) and 18:2(Δ9,15), were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2(Δ9,15) from endogenous 18:1(Δ9) when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop. PMID:24506492

  15. Microsomal Omega-3 Fatty Acid Desaturase Genes in Low Linolenic Acid Soybean Line RG10 and Validation of Major Linolenic Acid QTL

    PubMed Central

    Reinprecht, Yarmilla; Pauls, K. Peter

    2016-01-01

    High levels of linolenic acid (80 g kg−1) are associated with the development of off-flavors and poor stability in soybean oil. The development of low linolenic acid lines such as RG10 (20 g kg−1 linolenic acid) can reduce these problems. The level of linolenic acid in seed oil is determined by the activities of microsomal omega-3 fatty acid desaturases (FAD3). A major linolenic acid QTL (>70% of variation) on linkage group B2 (chromosome Gm14) was previously detected in a recombinant inbred line population from the RG10 × OX948 cross. The objectives of this study were to validate the major linolenic acid QTL in an independent population and characterize all the soybean FAD3 genes. Four FAD3 genes were sequenced and localized in RG10 and OX948 and compared to the genes in the reference Williams 82 genome. The FAD3A gene sequences mapped to the locus Glyma.14g194300 [on the chromosome Gm14 (B2)], which is syntenic to the FAD3B gene (locus Glyma.02g227200) on the chromosome Gm02 (D1b). The location of the FAD3A gene is the same as was previously determined for the fan allele, that conditions low linolenic acid content and several linolenic acid QTL, including Linolen 3-3, mapped previously with the RG10 × OX948 population and confirmed in the PI 361088B × OX948 population as Linolen-PO (FAD3A). The FAD3B gene-based marker, developed previously, was mapped to the chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL [Linolen-RO(FAD3B)] in the RG10 × OX948 genetic map and corresponds well with the in silico position of the FAD3B gene sequences. FAD3C and FAD3D gene sequences, mapped to syntenic regions on chromosomes Gm18 (locus Glyma.18g062000) and Gm11 (locus Glyma.11g227200), respectively. Association of linolenic acid QTL with the desaturase genes FAD3A and FAD3B, their validation in an independent population, and development of FAD3 gene-specific markers should simplify and accelerate breeding for low linolenic acid soybean

  16. Serum phospholipid monounsaturated fatty acid composition and Δ-9-desaturase activity are associated with early alteration of fasting glycemic status.

    PubMed

    Cho, Jae Sun; Baek, Seung Han; Kim, Ji Young; Lee, Jong Ho; Kim, Oh Yoen

    2014-09-01

    Because alterations in blood fatty acid (FA) composition by dietary lipids are associated with insulin resistance and related metabolic disorders, we hypothesized that serum phospholipid FA composition would reflect the early alteration of fasting glycemic status, even in people without metabolic syndrome (MetS). To examine this hypothesis, serum phospholipid FA, desaturase activities, fasting glycemic status, and cardiometabolic parameters were measured in study participants (n = 1022; 30-69 years; male, n = 527; female, n = 495; nondiabetics without disease) who were stratified into normal fasting glucose (NFG) and impaired fasting glucose (IFG) groups. Total monounsaturated FA (MUFA), oleic acid (OA; 18:1n-9), dihomo-γ-linolenic acid (DGLA; 20:3n-6), Δ-9-desaturase activity (D9D; 18:1n-9/18:0), and DGLA/linoleic acid (20:3n-6/18:2n-6) in serum phospholipids were significantly higher in IFG subjects than NFG controls. Study subjects were subdivided into 4 groups, based on fasting glucose levels and MetS status. Palmitoleic acid (16:1n-7) was highest in IFG-MetS and lowest in NFG-non-MetS subjects. Oleic acid and D9D were higher in IFG-MetS than in the other 3 groups. Dihomo-γ-linolenic acid and DGLA/linoleic acid were higher in MetS than in non-MetS, regardless of fasting glucose levels. The high-sensitivity C-reactive proteins (hs-CRPs) and 8-epi-prostaglandin-F2α were higher in IFG than in NFG, regardless of MetS status. Oxidized low-density lipoproteins were higher in IFG-MetS than in the other 3 groups. Total MUFAs, OA, and D9D were positively correlated with homeostasis model assessment of insulin resistance, fasting glucose, triglyceride, hs-CRP, and 8-epi-prostaglandin-F2α. Palmitoleic acid was positively correlated with triglyceride and hs-CRP. Lastly, total MUFA, OA, palmitoleic acid, and D9D were associated with early alteration of fasting glycemic status, therefore suggesting that these may be useful markers for predicting the risk of type 2

  17. Fatty acid desaturase 1 knockout mice are lean with improved glycemic control and decreased development of atheromatous plaque

    PubMed Central

    Powell, David R; Gay, Jason P; Smith, Melinda; Wilganowski, Nathaniel; Harris, Angela; Holland, Autumn; Reyes, Maricela; Kirkham, Laura; Kirkpatrick, Laura L; Zambrowicz, Brian; Hansen, Gwenn; Platt, Kenneth A; van Sligtenhorst, Isaac; Ding, Zhi-Ming; Desai, Urvi

    2016-01-01

    Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. PMID:27382320

  18. A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega13 desaturation in methylotrophic yeast and tobacco.

    PubMed

    Kajikawa, Masataka; Yamato, Katsuyuki T; Kohzu, Yoshito; Shoji, Shin-ichiro; Matsui, Keisuke; Tanaka, Yoshikazu; Sakai, Yasuyoshi; Fukuzawa, Hideya

    2006-01-01

    Pinolenic acid (PA; 18:3Delta(5,9,12)) and coniferonic acid (CA; 18:4Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Delta(9,12)) and alpha-linolenic acid (ALA; 18:3Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1Delta(9) but not 18:1Delta(11). Unexpectedly, CrDES also showed Delta(7)-desaturase activity on 20:2Delta(11,14) and 20:3Delta(11,14,17) to produce 20:3Delta(7,11,14) and 20:4Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega13' double bonds, these results indicate that CrDES has omega13 desaturase activity for omega9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale. PMID:16267098

  19. Stearoyl-CoA Desaturase-1: Is It the Link between Sulfur Amino Acids and Lipid Metabolism?

    PubMed Central

    Poloni, Soraia; Blom, Henk J.; Schwartz, Ida V. D.

    2015-01-01

    An association between sulfur amino acids (methionine, cysteine, homocysteine and taurine) and lipid metabolism has been described in several experimental and population-based studies. Changes in the metabolism of these amino acids influence serum lipoprotein concentrations, although the underlying mechanisms are still poorly understood. However, recent evidence has suggested that the enzyme stearoyl-CoA desaturase-1 (SCD-1) may be the link between these two metabolic pathways. SCD-1 is a key enzyme for the synthesis of monounsaturated fatty acids. Its main substrates C16:0 and C18:0 and products palmitoleic acid (C16:1) and oleic acid (C18:1) are the most abundant fatty acids in triglycerides, cholesterol esters and membrane phospholipids. A significant suppression of SCD-1 has been observed in several animal models with disrupted sulfur amino acid metabolism, and the activity of SCD-1 is also associated with the levels of these amino acids in humans. This enzyme also appears to be involved in the etiology of metabolic syndromes because its suppression results in decreased fat deposits (regardless of food intake), improved insulin sensitivity and higher basal energy expenditure. Interestingly, this anti-obesogenic phenotype has also been described in humans and animals with sulfur amino acid disorders, which is consistent with the hypothesis that SCD-1 activity is influenced by these amino acids, in particularly cysteine, which is a strong and independent predictor of SCD-1 activity and fat storage. In this narrative review, we discuss the evidence linking sulfur amino acids, SCD-1 and lipid metabolism. PMID:26046927

  20. Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding Δ12- and Δ15-fatty acid desaturases in Hansenula polymorpha.

    PubMed

    Sangwallek, Juthaporn; Kaneko, Yoshinobu; Tsukamoto, Tomoya; Marui, Makoto; Sugiyama, Minetaka; Ono, Hisayo; Bamba, Takeshi; Fukusaki, Eiichiro; Harashima, Satoshi

    2014-01-01

    Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha Δ12-fatty acid desaturase (HpFad2) and Δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, Δ9, Δ12) and α-linolenic acid (ALA, αC18:3, Δ9, Δ12, Δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215bp and 1239bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2Δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode Δ12- and Δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a Δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post

  1. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304

    PubMed Central

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-01-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  2. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304.

    PubMed

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-06-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  3. Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO).

    PubMed

    Huerlimann, Roger; Steinig, Eike J; Loxton, Heather; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2014-07-15

    Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω-3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ∆(6)-, ∆(8)-, ∆(5)- and ∆(4)-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made. PMID:24802118

  4. Synthesis and use of stereospecifically deuterated analogues of palmitic Acid to investigate the stereochemical course of the delta11 desaturase of the processionary moth.

    PubMed

    Abad, José-Luis; Villorbina, Gemma; Fabriàs, Gemma; Camps, Francisco

    2004-10-15

    Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the immediate sex pheromone precursor. In this article, we describe the synthesis of different stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher the stereochemical course of the T. pityocampa Delta(11) desaturase. The synthesis of probes has been carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental evidence is presented that the Delta(11) desaturase of T. pityocampa transforms palmitic acid into (Z)-11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12. PMID:15471459

  5. Susceptibility of Podocytes to Palmitic Acid Is Regulated by Stearoyl-CoA Desaturases 1 and 2

    PubMed Central

    Sieber, Jonas; Weins, Astrid; Kampe, Kapil; Gruber, Stefan; Lindenmeyer, Maja T.; Cohen, Clemens D.; Orellana, Jana M.; Mundel, Peter; Jehle, Andreas W.

    2014-01-01

    Type 2 diabetes mellitus is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are highly susceptible to saturated FFAs but not to protective, monounsaturated FFAs. We report that patients with diabetic nephropathy develop alterations in glomerular gene expression of enzymes involved in fatty acid metabolism, including induction of stearoyl-CoA desaturase (SCD)-1, which converts saturated to monounsaturated FFAs. By IHC of human renal biopsy specimens, glomerular SCD-1 induction was observed in podocytes of patients with diabetic nephropathy. Functionally, the liver X receptor agonists TO901317 and GW3965, two known inducers of SCD, increased Scd-1 and Scd-2 expression in cultured podocytes and reduced palmitic acid–induced cell death. Similarly, overexpression of Scd-1 attenuated palmitic acid–induced cell death. The protective effect of TO901317 was associated with a reduction of endoplasmic reticulum stress. It was lost after gene silencing of Scd-1/-2, thereby confirming that the protective effect of TO901317 is mediated by Scd-1/-2. TO901317 also shifted palmitic acid–derived FFAs into biologically inactive triglycerides. In summary, SCD-1 up-regulation in diabetic nephropathy may be part of a protective mechanism against saturated FFA-derived toxic metabolites that drive endoplasmic reticulum stress and podocyte death. PMID:23867797

  6. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  7. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

    PubMed Central

    He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  8. Relationship between a common variant in the fatty acid desaturase (FADS) cluster and eicosanoid generation in humans.

    PubMed

    Hester, Austin G; Murphy, Robert C; Uhlson, Charis J; Ivester, Priscilla; Lee, Tammy C; Sergeant, Susan; Miller, Leslie R; Howard, Timothy D; Mathias, Rasika A; Chilton, Floyd H

    2014-08-01

    Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B4, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases. PMID:24962583

  9. Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases.

    PubMed

    Kabeya, Naoki; Yamamoto, Yoji; Cummins, Scott F; Elizur, Abigail; Yazawa, Ryosuke; Takeuchi, Yutaka; Haga, Yutaka; Satoh, Shuichi; Yoshizaki, Goro

    2015-10-01

    To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via β-oxidation. PMID:26112824

  10. Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition

    PubMed Central

    Mohammadzadeh, Fatemeh; Mosayebi, Gholamali; Montazeri, Vahid; Darabi, Maryam; Fayezi, Shabnam; Shaaker, Maghsod; Rahmati, Mohammad; Baradaran, Behzad; Mehdizadeh, Amir

    2014-01-01

    Purpose Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. Methods Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. Results Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 µM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. Conclusion The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition

  11. Genetic variants of the fatty acid desaturase gene cluster are associated with plasma LDL cholesterol levels in Japanese males.

    PubMed

    Sone, Yasuko; Kido, Toshimi; Ainuki, Tomomi; Sonoda, Mariko; Ichi, Ikuyo; Kodama, Satoru; Sone, Hirohito; Kondo, Kazuo; Morita, Yutaka; Egawa, Shigenobu; Kawahara, Kazuo; Otsuka, Yuzuru; Fujiwara, Yoko

    2013-01-01

    Fatty acid (FA) compositions in tissues are related to metabolic disorders, and consequently the appropriate management of underlying FA compositions in tissues is considered to be important. However, the relationship among the serum lipid profiles, the FA composition of the red blood cell (RBC) membranes and genetic variations in the fatty acid desaturase (FADS) genes in Japanese men is unclear. In this study, the subjects recruited were 137 Japanese men, 40 to 60 y old, who had a regular health checkup. Their serum lipid profile and the relative FA composition of the RBC membranes were measured. They were genotyped for the single nucleotide polymorphisms (SNPs) rs174553, rs174546, rs99780 and rs174583 in FADS gene. Multiple regression analysis was conducted to detect the relationship among hyperlipidemia, the FA composition of the RBC and the FADS genotypes. As a result, the homozygous genotype for the minor alleles in rs174553, rs174546, rs99780 were found to be associated with lower low-density lipoprotein cholesterol (LDL-C) levels and a lower LDL-C/total-cholesterol ratio. The homozygous genotype for the minor alleles reduced the risk of high LDL-C level (R2=0.50, β=-0.20, p=0.009), whereas, the arachidonic acid (AA) levels in the carriers of the homozygous genotype for the minor alleles tended to be lower compared with the carriers of the major alleles. However, no significant differences were observed in any FA level among the three genotypes for four SNPs. These results indicate that the appropriate management of serum LDL-C levels depending on genetic predisposition in FADS genotypes should be encouraged. PMID:24064733

  12. Polymorphisms in Fatty Acid Desaturase (FADS) Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA) Supplementation

    PubMed Central

    Cormier, Hubert; Rudkowska, Iwona; Thifault, Elisabeth; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

    2013-01-01

    Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM). Polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS) and serum fatty acid composition. Objective: To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG) and fasting insulin (FI) responses following a 6-week n-3 polyunsaturated fatty acids (PUFA) supplementation. Methods: 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g–2.2 g of EPA + 1.1 g of DHA). Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03) was performed. Results: Carriers of the minor allele for rs482548 (FADS2) had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008). For FI levels, a genotype effect was observed with one SNP (rs174456). For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively). Conclusion: Results suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation. PMID:24705214

  13. CO-EXPRESSION OF THE BORAGE DELTA-6 DESATURASE AND THE ARABIDOPSIS DELTA-15 DESATURASE RESULTS IN HIGH ACCUMULATION OF STEARIDONIC ACID IN THE SEEDS OF TRANSGENIC SOYBEAN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two relatively rare fatty acids, gamma-linolenic acid (GLA) and stearidonic acid (STA) have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion e...

  14. Cancer cell dependence on unsaturated fatty acids implicates stearoyl-CoA desaturase as a target for cancer therapy.

    PubMed

    Roongta, Urvashi V; Pabalan, Jonathan G; Wang, Xinyu; Ryseck, Rolf-Peter; Fargnoli, Joseph; Henley, Benjamin J; Yang, Wen-Pin; Zhu, Jun; Madireddi, Malavi T; Lawrence, R Michael; Wong, Tai W; Rupnow, Brent A

    2011-11-01

    Emerging literature suggests that metabolic pathways play an important role in the maintenance and progression of human cancers. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. In the studies reported here, we aimed to understand whether tumor cells require the activity of either human isoform of stearoyl-CoA-desaturase (SCD1 or SCD5) for survival. Inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition, when tumor cells are cultured in reduced (2%) serum conditions, but has little impact on cells cultured in 10% serum. Depletion of SCD5 had minimal effects on cell growth or apoptosis. Consistent with the observed dependence on SCD1, but not SCD5, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids, the product of the enzymatic reaction catalyzed by SCD1. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that desaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy. PMID:21954435

  15. Dihydroceramide-desaturase-1-mediated caspase 9 activation through ceramide plays a pivotal role in palmitic acid-induced HepG2 cell apoptosis.

    PubMed

    Zhu, Qun; Yang, Jianjun; Zhu, Rongping; Jiang, Xin; Li, Wanlian; He, Songqing; Jin, Junfei

    2016-09-01

    In this study, results showed that the inhibition of PA-induced HepG2 cell growth takes place in a time- and concentration-dependent manner, that activation of caspase 9 is necessary for PA-induced HepG2 cell apoptosis, that dihydroceramide desaturase 1 (DES1) plays a key role in PA-mediated caspase 9 and caspase 3 activation, and that palmitoleic acid (POA), an omega-7 monounsaturated fatty acid, reverses PA-induced apoptosis through DES1 → Ceramide → Caspase 9 → Caspase 3 signaling. PMID:27364952

  16. Δ-6 Desaturase substrate competition: dietary linoleic acid (18:2n-6) has only trivial effects on α-linolenic acid (18:3n-3) bioconversion in the teleost rainbow trout.

    PubMed

    Emery, James A; Hermon, Karen; Hamid, Noor K A; Donald, John A; Turchini, Giovanni M

    2013-01-01

    It is generally accepted that, in vertebrates, omega-3 (n-3) and omega-6 (n-6) poly-unsaturated fatty acids (PUFA) compete for Δ-6 desaturase enzyme in order to be bioconverted into long-chain PUFA (LC-PUFA). However, recent studies into teleost fatty acid metabolism suggest that these metabolic processes may not conform entirely to what has been previously observed in mammals and other animal models. Recent work on rainbow trout has led us to question specifically if linoleic acid (LA, 18∶2n-6) and α-linolenic acid (ALA, 18∶3n-3) (Δ-6 desaturase substrates) are in direct competition for access to Δ-6 desaturase. Two experimental diets were formulated with fixed levels of ALA, while LA levels were varied (high and low) to examine if increased availability of LA would result in decreased bioconversion of ALA to its LC-PUFA products through substrate competition. No significant difference in ALA metabolism towards n-3 LC-PUFA was exhibited between diets while significant differences were observed in LA metabolism towards n-6 LC-PUFA. These results are evidence for minor if any competition between substrates for Δ-6 desaturase, suggesting that, paradoxically, the activity of Δ-6 desaturase on n-3 and n-6 substrates is independent. These results call for a paradigm shift in the way we approach teleost fatty acid metabolism. The findings are also important with regard to diet formulation in the aquaculture industry as they indicate that there should be no concern for possible substrate competition between 18∶3n-3 and 18∶2n-6, when aiming at increased n-3 LC-PUFA bioconversion in vivo. PMID:23460861

  17. bZIP67 Regulates the Omega-3 Fatty Acid Content of Arabidopsis Seed Oil by Activating FATTY ACID DESATURASE3[W][OPEN

    PubMed Central

    Mendes, Ana; Kelly, Amélie A.; van Erp, Harrie; Shaw, Eve; Powers, Stephen J.; Kurup, Smita; Eastmond, Peter J.

    2013-01-01

    Arabidopsis thaliana seed maturation is accompanied by the deposition of storage oil, rich in the essential ω-3 polyunsaturated fatty acid α-linolenic acid (ALA). The synthesis of ALA is highly responsive to the level of FATTY ACID DESATURASE3 (FAD3) expression, which is strongly upregulated during embryogenesis. By screening mutants in LEAFY COTYLEDON1 (LEC1)–inducible transcription factors using fatty acid profiling, we identified two mutants (lec1-like and bzip67) with a seed lipid phenotype. Both mutants share a substantial reduction in seed ALA content. Using a combination of in vivo and in vitro assays, we show that bZIP67 binds G-boxes in the FAD3 promoter and enhances FAD3 expression but that activation is conditional on bZIP67 association with LEC1-LIKE (L1L) and NUCLEAR FACTOR-YC2 (NF-YC2). Although FUSCA3 and ABSCISIC ACID INSENSITIVE3 are required for L1L and bZIP67 expression, neither protein is necessary for [bZIP67:L1L:NF-YC2] to activate FAD3. We conclude that a transcriptional complex containing L1L, NF-YC2, and bZIP67 is induced by LEC1 during embryogenesis and specifies high levels of ALA production for storage oil by activating FAD3 expression. PMID:23995083

  18. Computer Simulation Model for the Biosynthesis of Galactosyldiacylglycerols and Fatty Acid Desaturation in Plants (Determination of Rates of Desaturase Activity in Monogalactosyldiacylglycerol).

    PubMed Central

    Williams, J. P.; Khan, M. U.; Wong, D.

    1993-01-01

    The level of unsaturation of the constituent fatty acids of many glycerolipids in plant membranes is modified by environmental factors. The measurement of the rate of the desaturation of these fatty acids is essential to an understanding of how plants adapt to changing environments. This is difficult because of the complexity of the system and the problems involved in measuring rates of these enzyme reactions in cell-free preparations. A computer program has been developed that simulates the synthesis of galactosyldiacylglycerols and desaturation of their fatty acids in chloroplasts. The program uses the rate of incorporation and distribution of 14C in fatty acids after 14CO2 feeding to estimate rates of desaturation in the fatty acids of glycerolipids. Data are presented to demonstrate the use of the program in comparing rates of desaturation in the five enzyme reactions associated with monogalactosyldiacylglycerol in the chloroplastic pathway of leaves from Brassica napus. The method represents a quick, reliable, and accurate measure of desaturase activity in vivo and is the only method available to estimate desaturase activity of all five enzymes at the same time. PMID:12231750

  19. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  20. Omega-3 fatty acid deficiency selectively up-regulates delta6-desaturase expression and activity indices in rat liver: prevention by normalization of omega-3 fatty acid status.

    PubMed

    Hofacer, Rylon; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Magrisso, I Jack; Benoit, Stephen C; McNamara, Robert K

    2011-09-01

    This study investigated the effects of perinatal dietary omega-3 (n-3) fatty acid depletion and subsequent repletion on the expression of genes that regulate long-chain (LC) polyunsaturated fatty acid biosynthesis in rat liver and brain. It was hypothesized that chronic n-3 fatty acid deficiency would increase liver Fads1 and Fads2 messenger RNA (mRNA) expression/activity and that n-3 fatty acid repletion would normalize this response. Adult rats fed the n-3-free diet during perinatal development exhibited significantly lower erythrocyte, liver, and frontal cortex LCn-3 fatty acid composition and reciprocal elevations in LC omega-6 (n-6) fatty acid composition compared with controls (CONs) and repleted rats. Liver Fads2, but not Fads1, Elovl2, or Elovl5, mRNA expression was significantly greater in n-3-deficient (DEF) rats compared with CONs and was partially normalized in repleted rats. The liver 18:3n-6/18:2n-6 ratio, an index of delta6-desturase activity, was significantly greater in DEF rats compared with CON and repleted rats and was positively correlated with Fads2 mRNA expression among all rats. The liver 18:3n-6/18:2n-6 ratio, but not Fads2 mRNA expression, was also positively correlated with erythrocyte and frontal cortex LCn-6 fatty acid compositions. Neither Fads1 or Fads2 mRNA expression was altered in brain cortex of DEF rats. These results confirm previous findings that liver, but not brain, delta6-desaturase expression and activity indices are negatively regulated by dietary n-3 fatty acids. PMID:22024496

  1. Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil.

    PubMed

    Li, Xueyuan; Mei, Desheng; Liu, Qing; Fan, Jing; Singh, Surinder; Green, Allan; Zhou, Xue-Rong; Zhu, Li-Hua

    2016-01-01

    High oleic oil is an important industrial feedstock that has been one of the main targets for oil improvement in a number of oil crops. Crambe (Crambe abyssinica) is a dedicated oilseed crop, suitable for industrial oil production. In this study, we down-regulated the crambe fatty acid desaturase (FAD) and fatty acid elongase (FAE) genes for creating high oleic seed oil. We first cloned the crambe CaFAD2, CaFAD3 and CaFAE1 genes. Multiple copies of each of these genes were isolated, and the highly homologous sequences were used to make RNAi constructs. These constructs were first tested in Arabidopsis, which led to the elevated oleic or linoleic levels depending on the genes targeted, indicating that the RNAi constructs were effective in regulating the expression of the target genes in nonidentical but closely related species. Furthermore, down-regulation of CaFAD2 and CaFAE1 in crambe with the FAD2-FAE1 RNAi vector resulted in even more significant increase in oleic acid level in the seed oil with up to 80% compared to 13% for wild type. The high oleic trait has been stable in subsequent five generations and the GM line grew normally in greenhouse. This work has demonstrated the great potential of producing high oleic oil in crambe, thus contributing to its development into an oil crop platform for industrial oil production. PMID:25998013

  2. A Functional Variant in the Stearoyl-CoA Desaturase Gene Promoter Enhances Fatty Acid Desaturation in Pork

    PubMed Central

    Estany, Joan; Ros-Freixedes, Roger; Tor, Marc; Pena, Ramona N.

    2014-01-01

    There is growing public concern about reducing saturated fat intake. Stearoyl-CoA desaturase (SCD) is the lipogenic enzyme responsible for the biosynthesis of oleic acid (18∶1) by desaturating stearic acid (18∶0). Here we describe a total of 18 mutations in the promoter and 3′ non-coding region of the pig SCD gene and provide evidence that allele T at AY487830:g.2228T>C in the promoter region enhances fat desaturation (the ratio 18∶1/18∶0 in muscle increases from 3.78 to 4.43 in opposite homozygotes) without affecting fat content (18∶0+18∶1, intramuscular fat content, and backfat thickness). No mutations that could affect the functionality of the protein were found in the coding region. First, we proved in a purebred Duroc line that the C-T-A haplotype of the 3 single nucleotide polymorphisms (SNPs) (g.2108C>T; g.2228T>C; g.2281A>G) of the promoter region was additively associated to enhanced 18∶1/18∶0 both in muscle and subcutaneous fat, but not in liver. We show that this association was consistent over a 10-year period of overlapping generations and, in line with these results, that the C-T-A haplotype displayed greater SCD mRNA expression in muscle. The effect of this haplotype was validated both internally, by comparing opposite homozygote siblings, and externally, by using experimental Duroc-based crossbreds. Second, the g.2281A>G and the g.2108C>T SNPs were excluded as causative mutations using new and previously published data, restricting the causality to g.2228T>C SNP, the last source of genetic variation within the haplotype. This mutation is positioned in the core sequence of several putative transcription factor binding sites, so that there are several plausible mechanisms by which allele T enhances 18∶1/18∶0 and, consequently, the proportion of monounsaturated to saturated fat. PMID:24465944

  3. Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

    SciTech Connect

    Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; Inmon, Jay J.; Millhollon, Jon C.; Liechty, Zach; Page, Justin T.; Jenks, Matthew A.; Chapman, Kent D.; Udall, Joshua A.; Gore, Michael A.; Dyer, John M.

    2014-11-18

    The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level

  4. CsSAD: a fatty acid desaturase gene involved in abiotic resistance in Camellia sinensis (L.).

    PubMed

    Ding, Z T; Shen, J Z; Pan, L L; Wang, Y U; Li, Y S; Wang, Y; Sun, H W

    2016-01-01

    Tea (Camellia sinensis L.) is a thermophilic evergreen woody plant that has poor cold tolerance. The SAD gene plays a key role in regulating fatty acid synthesis and membrane lipid fluidity in response to temperature change. In this study, full-length SAD cDNA was cloned from tea leaves using rapid amplification of cDNA ends and polymerase chain reaction (PCR)-based methods. Sequence analysis demonstrated that CsSAD had a high similarity to other corresponding cDNAs. At 25°C, the CsSAD transcriptional level was highest in the leaf and lowest in the stem, but there was no obvious difference between the root and stem organs. CsSAD expression was investigated by reverse transcription-PCR, which showed that CsSAD was upregulated at 4° and -5°C. At 25°C, CsSAD was induced by polyethylene glycol, abscisic acid, and wounding, and a similar trend was observed at 4°C, but the mean expression level at 4°C was lower than that at 25°C. Under natural cold acclimation, the 'CsCr05' variety's CsSAD expression level increased before decreasing. The CsSAD expression level in variety 'CsCr06' showed no obvious change at first, but rapidly increased to a maximum when the temperature was very low. Our study demonstrates that CsSAD is upregulated in response to different abiotic conditions, and that it is important to study the stress resistance of the tea plant, particularly in response to low temperature, drought, and wounding. PMID:26985937

  5. Stacking of a stearoyl-ACP thioesterase with a dual-silenced palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean.

    PubMed

    Park, Hyunwoo; Graef, George; Xu, Yixiang; Tenopir, Patrick; Clemente, Tom E

    2014-10-01

    Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end-use applications. However, the hydrogenation process leads to the formation of trans-fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl-ACP thioesterase into soybean and the subsequent stacking with an event that is dual-silenced in palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase expression in a seed-specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl-ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%-19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%-40%, while the stearic level remained elevated. PMID:24909647

  6. Conferring high-temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene.

    PubMed

    Nakamura, Shinya; Hondo, Kana; Kawara, Tomoko; Okazaki, Yozo; Saito, Kazuki; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

    2016-02-01

    We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks. PMID:26132723

  7. ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 Are Responsible for Making Omega-7 Fatty Acids in the Arabidopsis Aleurone.

    PubMed

    Bryant, Fiona M; Munoz-Azcarate, Olaya; Kelly, Amélie A; Beaudoin, Frédéric; Kurup, Smita; Eastmond, Peter J

    2016-09-01

    Omega-7 monounsaturated fatty acids (ω-7s) are specifically enriched in the aleurone of Arabidopsis (Arabidopsis thaliana) seeds. We found significant natural variation in seed ω-7 content and used a Multiparent Advanced Generation Inter-Cross population to fine-map a major quantitative trait loci to a region containing ACYL-ACYL CARRIER PROTEIN DESATURASE1 (AAD1) and AAD3 We found that AAD3 expression is localized to the aleurone where mutants show an approximately 50% reduction in ω-7 content. By contrast, AAD1 is localized to the embryo where mutants show a small reduction in ω-9 content. Enzymatic analysis has previously shown that AAD family members possess both stearoyl- and palmitoyl-ACP Δ(9) desaturase activity, including the predominant isoform SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2. However, aad3 ssi2 aleurone contained the same amount of ω-7s as aad3 Within the AAD family, AAD3 shares the highest degree of sequence similarity with AAD2 and AAD4. Mutant analysis showed that AAD2 also contributes to ω-7 production in the aleurone, and aad3 aad2 exhibits an approximately 85% reduction in ω-7s Mutant analysis also showed that FATTY ACID ELONGASE1 is required for the production of very long chain ω-7s in the aleurone. Together, these data provide genetic evidence that the ω-7 pathway proceeds via Δ(9) desaturation of palmitoyl-ACP followed by elongation of the product. Interestingly, significant variation was also identified in the ω-7 content of Brassica napus aleurone, with the highest level detected being approximately 47% of total fatty acids. PMID:27462083

  8. Δ(9) desaturase protein expression and fatty acid composition of longissimus dorsi muscle in lambs fed green herbage or concentrate with or without added tannins.

    PubMed

    Vasta, Valentina; Priolo, Alessandro; Scerra, Manuel; Hallett, Katharine G; Wood, Jeffrey D; Doran, Olena

    2009-07-01

    The aims of this study were to investigate the effect of feeding system and of supplementation of tannins (8.93% DM) on the relationship between intramuscular fat content, fatty acid composition and Δ(9)desaturase (Δ(9)d) protein expression in longissimus dorsi muscle of lamb. Twenty-eight Comisana lambs (age 45days) were fed either vetch (Vicia sativa) or concentrate. The herbage diet was (i) lower in saturated fatty acids (especially in C16:0), C18:1 n-9 and in C18:2 n-6; (ii) higher in C16:1 and C18:3 n-3 when compared to concentrate. Within each feeding system the lambs were divided into two sub-groups, one of which received the diet without tannins supplementation, and the other was fed the diets supplemented with the tannins from Quebracho (Schinopsis lorentzii). The animals were slaughtered at age 105days. The concentrate feeding system increased (p<0.01) the total intramuscular fat content and the amount of SFA, MUFA and n-6 PUFA and decreased the level of n-3 PUFA (p=0.05) when compared to the vetch-fed animals but did not affect Δ(9) desaturase protein expression. There was no correlation between Δ(9)d protein expression and total intramuscular fatty acids, CLA and MUFA level. It was suggested that in ruminants, in contrast to monogastric animals, Δ(9)d expression does not play the key role in intramuscular fatty acids formation. Tannins supplementation resulted in higher (p<0.05) muscle levels of transC18:1 and C18:2 n-6. It has also increased Δ(9)d expression in the case of herbage-based diet but not in the case of concentrate-based diet. The mechanism of tannins action on the enzyme expression needs to be elucidated. PMID:20416712

  9. Loss of Function of FATTY ACID DESATURASE7 in Tomato Enhances Basal Aphid Resistance in a Salicylate-Dependent Manner1[W][OA

    PubMed Central

    Avila, Carlos A.; Arévalo-Soliz, Lirio M.; Jia, Lingling; Navarre, Duroy A.; Chen, Zhaorigetu; Howe, Gregg A.; Meng, Qing-Wei; Smith, Jonathon E.; Goggin, Fiona L.

    2012-01-01

    We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families. PMID:22291202

  10. Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.

    PubMed

    Radovanovic, Natasa; Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Cloutier, Sylvie

    2014-07-01

    With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed. PMID:24522837

  11. Expression analysis identifies FAD2-2 as the olive oleate desaturase gene mainly responsible for the linoleic acid content in virgin olive oil.

    PubMed

    Hernández, M Luisa; Padilla, María N; Mancha, Manuel; Martínez-Rivas, José M

    2009-07-22

    The effect of ripening stage and water regimen on oleate desaturase gene expression levels in the fruit of different olive ( Olea europaea L.) varieties was investigated to elucidate the contribution of each to the linoleic acid content in virgin olive oil. To this end, fatty acid analysis and quantitative real time PCR were performed using distinct olive tissues and different developmental stages from the Picual and Arbequina cultivars. The results showed that the olive FAD2-1, FAD2-2, and FAD6 genes were spatial and temporally regulated. In addition, the data indicated that FAD2-2 seems to be the main gene responsible for the linoleic acid content in the olive fruit mesocarp tissue. This conclusion was also confirmed when the study was extended to Hojiblanca, Picudo, and Manzanilla varieties. With regard to the water regimen, unlike the Picual cultivar, a small increase of linoleic acid was observed in the Arbequina variety cultivated with irrigation, which correlated well with the increase detected for the FAD2-2 gene expression level. All of these data strongly suggest that FAD2-2 is the main gene that determines the linoleic acid content in the virgin olive oil. PMID:19601663

  12. Transgenic Expression of n-3 Fatty Acid Desaturase (fat-1) in C57/BL6 Mice: Effects on Glucose Homeostasis and Body Weight

    PubMed Central

    Ji, Shaonin; Hardy, Robert W.; Wood, Philip A.

    2009-01-01

    The fat-1 gene, derived from C. elegans, encodes for a fatty acid n-3 desaturase. In order to study the potential metabolic benefits of n-3 fatty acids, independent of dietary fatty acids, we developed 7 lines of fat-1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein-2 (aP2) gene for adipocyte specific expression (AP-lines). We were unable to obtain homozygous fat-1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat-1 transgenic mice (AP-3) fed a high n-6 unsaturated fat (HUSF) diet had an n-6/n-3 fatty acid ratio reduced by 23% (p< 0.025) and the n-3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (p< 0.020). Docosahexaenoic acid (DHA) was increased by 19 % (p< 0.015) in white adipose tissue. Male AP-3-fat-1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high-carbohydrate (HC) diet. In contrast, the female AP-3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat-1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat-1 mice have reduced glucose tolerance compared to wild-type mice. Finally the inability of fat-1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n-3 fatty acids may be detrimental to reproduction. PMID:19396841

  13. Umbilical cord PUFA are determined by maternal and child fatty acid desaturase (FADS) genetic variants in the Avon Longitudinal Study of Parents and Children (ALSPAC).

    PubMed

    Lattka, Eva; Koletzko, Berthold; Zeilinger, Sonja; Hibbeln, Joseph R; Klopp, Norman; Ring, Susan M; Steer, Colin D

    2013-04-14

    Fetal supply with long-chain PUFA (LC-PUFA) during pregnancy is important for brain growth and visual and cognitive development and is provided by materno-fetal placental transfer. We recently showed that maternal fatty acid desaturase (FADS) genotypes modulate the amounts of LC-PUFA in maternal blood. Whether FADS genotypes influence the amounts of umbilical cord fatty acids has not been investigated until now. The aim of the present study was to investigate the influence of maternal and child FADS genotypes on the amounts of LC-PUFA in umbilical cord venous plasma as an indicator of fetal fatty acid supply during pregnancy. A total of eleven cord plasma n-6 and n-3 fatty acids were analysed for association with seventeen FADS gene cluster SNP in over 2000 mothers and children from the Avon Longitudinal Study of Parents and Children. In a multivariable analysis, the maternal genotype effect was adjusted for the child genotype and vice versa to estimate which of the two has the stronger influence on cord plasma fatty acids. Both maternal and child FADS genotypes and haplotypes influenced amounts of cord plasma LC-PUFA and fatty acid ratios. Specifically, most analysed maternal SNP were associated with cord plasma levels of the precursor n-6 PUFA, whereas the child genotypes were mainly associated with more highly desaturated n-6 LC-PUFA. This first study on FADS genotypes and cord fatty acids suggests that fetal LC-PUFA status is determined to some extent by fetal fatty acid conversion. Associations of particular haplotypes suggest specific effects of SNP rs498793 and rs968567 on fatty acid metabolism. PMID:22877655

  14. Association between polymorphisms in the fatty acid desaturase gene cluster and the plasma triacylglycerol response to an n-3 PUFA supplementation.

    PubMed

    Cormier, Hubert; Rudkowska, Iwona; Paradis, Ann-Marie; Thifault, Elisabeth; Garneau, Véronique; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

    2012-08-01

    Eicosapentaenoic and docosahexaenoic acids have been reported to have a variety of beneficial effects on cardiovascular disease risk factors. However, a large inter-individual variability in the plasma lipid response to an omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation is observed in different studies. Genetic variations may influence plasma lipid responsiveness. The aim of the present study was to examine the effects of a supplementation with n-3 PUFA on the plasma lipid profile in relation to the presence of single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster. A total of 208 subjects from Quebec City area were supplemented with 3 g/day of n-3 PUFA, during six weeks. In a statistical model including the effect of the genotype, the supplementation and the genotype by supplementation interaction, SNP rs174546 was significantly associated (p = 0.02) with plasma triglyceride (TG) levels, pre- and post-supplementation. The n-3 supplementation had an independent effect on plasma TG levels and no significant genotype by supplementation interaction effects were observed. In summary, our data support the notion that the FADS gene cluster is a major determinant of plasma TG levels. SNP rs174546 may be an important SNP associated with plasma TG levels and FADS1 gene expression independently of a nutritional intervention with n-3 PUFA. PMID:23016130

  15. Microbial production of dihomo-γ-linolenic acid by Δ5-desaturase gene-disruptants of Mortierella alpina 1S-4.

    PubMed

    Kikukawa, Hiroshi; Sakuradani, Eiji; Ando, Akinori; Okuda, Tomoyo; Shimizu, Sakayu; Ogawa, Jun

    2016-07-01

    We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4. PMID:26777235

  16. Fatty Acid Profiles and Stearoyl-CoA Desaturase Gene Expression in Longissimus dorsi Muscle of Growing Lambs Influenced by Addition of Tea Saponins and Soybean Oil

    PubMed Central

    Mao, H. L.; Wang, J. K.; Lin, J.; Liu, J. X.

    2012-01-01

    This study was conducted to determine the effects of dietary addition of tea saponins (TS) and soybean oil (SO) on fatty acid profile and gene expression of stearoyl-CoA desaturase (SCD) in longissimus dorsi (LD) muscle of growing lambs. Thirty-two Huzhou lambs were assigned to four dietary treatments in a 2×2 factorial arrangement with main effects of TS (0 or 3 g/d) and SO (0 or 30 g/kg of diet DM). The diet without additives was considered as NTNS (no TS or SO). After a feeding trial for 60 d, four lambs of each treatment were slaughtered to collect the samples of LD muscle. Percentage of trans-11 vaccenic acid was enhanced (p<0.05) in muscle of lambs fed TS and SO. The proportion of total conjugated linoleic acid (CLA) was increased (p<0.05) by SO, but decreased (p<0.05) by TS in LD muscle. The percentage of total saturated fatty acids in muscle was decreased (p<0.05) by addition of TS and SO, while addition of SO increased (p<0.05) the percentage of total polyunsaturated fatty acids. The ratio of cis-9, trans-11 CLA to tran-11 vaccenic acid was decreased (p<0.05) by TS, but increased (p<0.05) by SO. The same effects were observed in SCD mRNA expression. From these results it is indicated that including TS and SO in the diet of growing lambs affect the fatty acid profiles of LD muscle and that the proportion of cis-9, trans-11 CLA in the muscle influenced by TS and SO may be related to the SCD gene expression. PMID:25049609

  17. Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba

    PubMed Central

    2013-01-01

    Background Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. Results Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. Conclusions Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished

  18. Sequence variation determining stereochemistry of a Δ11 desaturase active in moth sex pheromone biosynthesis.

    PubMed

    Ding, Bao-Jian; Carraher, Colm; Löfstedt, Christer

    2016-07-01

    A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution. PMID:27163509

  19. Characterization of the liver X receptor-dependent regulatory mechanism of goat stearoyl-coenzyme A desaturase 1 gene by linoleic acid.

    PubMed

    Yao, D W; Luo, J; He, Q Y; Li, J; Wang, H; Shi, H B; Xu, H F; Wang, M; Loor, J J

    2016-05-01

    Stearoyl-coenzyme A desaturase 1 (SCD1) is a key enzyme in the biosynthesis of palmitoleic and oleic acid. Although the transcriptional regulatory mechanism of SCD1 via polyunsaturated fatty acids (PUFA) has been extensively explored in nonruminants, the existence of such mechanism in ruminant mammary gland remains unknown. In this study, we used goat genomic DNA to clone and sequence a 1,713-bp fragment of the SCD1 5' flanking region. Deletion assays revealed a core region of the promoter located between -415 and -109 bp upstream of the transcription start site, and contained the highly conserved PUFA response region. An intact PUFA response region was required for the basal transcriptional activity of SCD1. Linoleic acid reduced endogenous expression of SCD1 and sterol regulatory element binding factor-1 (SREBF1) in goat mammary epithelial cells. Further analysis indicated that both the sterol response element (SRE) and the nuclear factor Y (NF-Y) binding site in the SCD1 promoter were responsible for the inhibition effect by linoleic acid, whereas the effect was abrogated once NF-Y was deleted. In addition, SRE and NF-Y were partly responsible for the transcriptional activation induced via the liver X receptor agonist T 4506585 (Sigma-Aldrich, St. Louis, MO). When goat mammary epithelial cells were cultured with linoleic acid, addition of T 4506585 markedly increased SCD1 transcription in controls, but had no effect on cells with a deleted SRE promoter. These results demonstrated that linoleic acid can regulate SCD1 expression at the transcriptional level through SRE and NF-Y in a liver X receptor-dependent fashion in the goat mammary gland. PMID:26947306

  20. Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7.

    PubMed

    Lu, Yandu; Chi, Xiaoyuan; Yang, Qingli; Li, Zhaoxin; Liu, Shaofang; Gan, Qinhua; Qin, Song

    2009-11-01

    The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15 degrees C and 3.9-fold at 4 degrees C compared to the alga at 25 degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7. PMID:19728010

  1. The Green Microalga Chlamydomonas reinhardtii Has a Single ω-3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids1[C][W

    PubMed Central

    Nguyen, Hoa Mai; Cuiné, Stéphan; Beyly-Adriano, Audrey; Légeret, Bertrand; Billon, Emmanuelle; Auroy, Pascaline; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2013-01-01

    The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids. PMID:23958863

  2. Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4

    PubMed Central

    Kawashima, H.; Nishihara, M.; Hirano, Y.; Kamada, N.; Akimoto, K.; Konishi, K.; Shimizu, S.

    1997-01-01

    Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter). PMID:16535598

  3. Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4.

    PubMed

    Kawashima, H; Nishihara, M; Hirano, Y; Kamada, N; Akimoto, K; Konishi, K; Shimizu, S

    1997-05-01

    Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter). PMID:16535598

  4. Mutations in a novel 9-stearoyl-ACP-desaturase gene are associated with enhanced stearic acid levels in soybean seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2-4 % of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process...

  5. Insertion-deletions in a FADS2 intron 1 conserved regulatory locus control expression of fatty acid desaturases 1 and 2 and modulate response to simvastatin.

    PubMed

    Reardon, Holly T; Zhang, Jimmy; Kothapalli, Kumar S D; Kim, Andrea J; Park, Woo Jung; Brenna, J Thomas

    2012-07-01

    The fatty acid desaturase genes (FADS1 and FADS2) code for enzymes required for synthesis of omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFA) important in the central nervous system, inflammatory response, and cardiovascular health. SNPs in these genes are associated with numerous health outcomes, but it is unclear how genetic variation affects enzyme function. Here, lymphoblasts obtained from Japanese participants in the International HapMap Project were evaluated for association of expression microarray results with SNPs in the FADS gene cluster. Six SNPs in the first intron of the FADS2 gene were associated with FADS1 expression. A 10-SNP haplotype in FADS2 (rs2727270 to rs2851682) present in 24% of the population was associated with lower expression of FADS1. A highly conserved region coinciding with the most significant SNPs contained predicted binding sites for SREBP and PPARγ. Lymphoblasts homozygous for either the major or minor haplotype were treated with agonists for these transcription factors and expression of FADS1 and FADS2 determined. Simvastatin and the LXR agonist GW3965 both upregulated expression of FADS1 and FADS2; no response was found for PPARγ agonist rosiglitazone. The minor haplotype homozygotes had 20-40% higher induction of FADS1 and FADS2 after simvastatin or GW3965 treatment. A 22 bp polymorphic insertion-deletion (INDEL) was found 137 bp downstream from the putative sterol response element, as well as a 3 or 1 bp INDEL 81-83 bp downstream. All carriers of the minor haplotype had deletions while all carriers of the major haplotype had insertions. Individuals carrying the minor haplotype may be vulnerable to alterations in diet that reduce LCPUFA intake, and especially responsive to statin or marine oil therapy. PMID:22748975

  6. Isolation and characterization of a Delta5-desaturase from Oblongichytrium sp.

    PubMed

    Kumon, Yasuyuki; Kamisaka, Yasushi; Tomita, Nao; Kimura, Kazuyoshi; Uemura, Hiroshi; Yokochi, Toshihiro; Yokoyama, Rinka; Honda, Daiske

    2008-08-01

    We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Delta5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Delta5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms. PMID:18685196

  7. Aryl Hydrocarbon Receptor–Mediated Induction of Stearoyl-CoA Desaturase 1 Alters Hepatic Fatty Acid Composition in TCDD-Elicited Steatosis

    PubMed Central

    Angrish, Michelle M.; Jones, A.D.; Harkema, Jack R.; Zacharewski, Timothy R.

    2011-01-01

    2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) induces hepatic dyslipidemia mediated by the aryl hydrocarbon receptor (AhR). Stearoyl-CoA desaturase 1 (Scd1) performs the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis, desaturating 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. To further examine the role of Scd1 in TCDD-induced hepatotoxicity, comparative studies were performed in Scd1+/+ and Scd1−/− mice treated with 30 μg/kg TCDD. TCDD induced Scd1 activity, protein, and messenger RNA (mRNA) levels approximately twofold. In Scd1+/+ mice, hepatic effects were marked by increased vacuolization and inflammation and a 3.5-fold increase in serum alanine aminotransferase (ALT) levels. Hepatic triglycerides (TRGs) were induced 3.9-fold and lipid profiling by gas chromatography-mass spectroscopy measured a 1.9-fold increase in fatty acid (FA) levels, consistent with the induction of lipid transport genes. Induction of Scd1 altered FA composition by decreasing saturated fatty acid (SFA) molar ratios 8% and increasing MUFA molar ratios 9%. Furthermore, ChIP-chip analysis revealed AhR enrichment (up to 5.7-fold), and computational analysis identified 16 putative functional dioxin response elements (DREs) within Scd1 genomic loci. Band shift assays confirmed AhR binding with select DREs. In Scd1−/− mice, TCDD induced minimal hepatic vacuolization and inflammation, while serum ALT levels remained unchanged. Although Scd1 deficiency attenuated TCDD-induced TRG accumulation, overall FA levels remained unchanged compared with Scd1+/+ mice. In Scd1−/− mice, TCDD induced SFA ratios 8%, reduced MUFA ratios 13%, and induced polyunsaturated fatty acid ratios 5% relative to treated Scd1+/+ mice. Collectively, these results suggest that AhR regulation of Scd1 not only alters lipid composition but also contributes to the hepatotoxicity of TCDD. PMID:21890736

  8. An insect with a delta-12 desaturase, the jewel wasp Nasonia vitripennis, benefits from nutritional supply with linoleic acid.

    PubMed

    Brandstetter, Birgit; Ruther, Joachim

    2016-06-01

    The availability of linoleic acid (LA; C18:2(∆9,12)) is pivotal for animals. While vertebrates depend on a nutritional supply, some invertebrates, including the parasitic wasp Nasonia vitripennis, are able to synthesize LA from oleic acid (OA; C18:1(∆9)). This raises the question as to whether these animals nevertheless benefit from the additional uptake of LA with the diet. LA plays an important role in the sexual communication of N. vitripennis because males use it as a precursor for the synthesis of an abdominal sex pheromone attracting virgin females. We reared hosts of N. vitripennis that were fed diets enriched in the availability of stearic acid (SA: C18:0), OA or LA. N. vitripennis males developing on the different host types clearly differed in both the fatty acid composition of their body fat and sex pheromone titres. Males from LA-enriched hosts had an almost fourfold higher proportion of LA and produced significantly more sex pheromone than males from SA (2.2-fold) and OA (1.4-fold) enriched hosts, respectively. Our study demonstrates that animals being able to synthesize important nutrients de novo may still benefit from an additional supply with their diet. PMID:27116611

  9. An insect with a delta-12 desaturase, the jewel wasp Nasonia vitripennis, benefits from nutritional supply with linoleic acid

    NASA Astrophysics Data System (ADS)

    Brandstetter, Birgit; Ruther, Joachim

    2016-06-01

    The availability of linoleic acid (LA; C18:2∆9,12) is pivotal for animals. While vertebrates depend on a nutritional supply, some invertebrates, including the parasitic wasp Nasonia vitripennis, are able to synthesize LA from oleic acid (OA; C18:1∆9). This raises the question as to whether these animals nevertheless benefit from the additional uptake of LA with the diet. LA plays an important role in the sexual communication of N. vitripennis because males use it as a precursor for the synthesis of an abdominal sex pheromone attracting virgin females. We reared hosts of N. vitripennis that were fed diets enriched in the availability of stearic acid (SA: C18:0), OA or LA. N. vitripennis males developing on the different host types clearly differed in both the fatty acid composition of their body fat and sex pheromone titres. Males from LA-enriched hosts had an almost fourfold higher proportion of LA and produced significantly more sex pheromone than males from SA (2.2-fold) and OA (1.4-fold) enriched hosts, respectively. Our study demonstrates that animals being able to synthesize important nutrients de novo may still benefit from an additional supply with their diet.

  10. Characterization of the promoter and 5'-UTR intron of oleic acid desaturase (FAD2) gene in Brassica napus.

    PubMed

    Xiao, Gang; Zhang, Zhen Qian; Yin, Chang Fa; Liu, Rui Yang; Wu, Xian Meng; Tan, Tai Long; Chen, She Yuan; Lu, Chang Ming; Guan, Chun Yun

    2014-07-15

    In the present study, we characterized the transcriptional regulatory region (KF038144) controlling the expression of a constitutive FAD2 in Brassica napus. There are multiple FAD2 gene copies in B. napus genome. The FAD2 gene characterized and analyzed in the study is located on chromosome A5 and was designated as BnFAD2A5-1. BnFAD2A5-1 harbors an intron (1,192 bp) within its 5'-untranslated region (5'-UTR). This intron demonstrated promoter activity. Deletion analysis of the BnFAD2A5-1 promoter and intron through the β-glucuronidase (GUS) reporter system revealed that the -220 to -1 bp is the minimum promoter region, while -220 to -110 bp and +34 to +285 bp are two important regions conferring high-levels of transcription. BnFAD2 transcripts were induced by light, low temperature, and abscisic acid (ABA). These observations demonstrated that not only the promoter but also the intron are involved in controlling the expression of the BnFAD2A5-1 gene. The intron-mediated regulation is an essential aspect of the gene expression regulation. PMID:24811682

  11. Multiple acyl-CoA desaturase-encoding transcripts in pheromone glands of Helicoverpa assulta, the oriental tobacco budworm.

    PubMed

    Jeong, Seong Eun; Rosenfield, Claire-Lise; Marsella-Herrick, Patricia; Man You, Kyung; Knipple, Douglas C

    2003-06-01

    Seven desaturase cDNAs were isolated from pheromone glands of Helicoverpa assulta, a moth producing a sex pheromone blend with high Z9-16:Ald and low Z11-16:Ald, opposite to what is found in other heliothine moths such as Helicoverpa zea. Six of the seven sequences map onto recently defined lepidopteran desaturase sequence lineages and the other is orthologous to a desaturase sequence previously reported only in H. zea. The levels of desaturase-encoding transcripts in pheromone glands were determined and the three most abundant ones were functionally expressed in a desaturase-deficient mutant strain of Saccharomyces cerevisiae. The HassNPVE transcript, shown to encode a delta9 desaturase producing more Z9-18:Acid than Z9-16:Acid, was the most abundant, followed by the HassKPSE transcript, shown to encode a delta9 desaturase producing more Z9-16:Acid than Z9-18:Acid, and by the HassLPAQ transcript, shown to encode a delta11 desaturase producing only Z11-16:Acid. Thus, the relative amounts of transcripts encoding two delta9 desaturases and a single delta11 desaturase in H. assulta pheromone glands were consistent with the relative amounts of unsaturated fatty acid precursors required to produce the major and minor sex pheromone components of this species. Desaturase transcript levels in pheromone glands were also found to be as high during scotophase as during light phase, when pheromone production ceases. The other four transcripts were present at extremely low levels in H. assulta pheromone glands and the functional roles of their encoded desaturase-homologous proteins could not be determined. PMID:12770579

  12. Delta-9 desaturase from sharpshooters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic analyses of several leafhoppers identified the first leafhopper delta-9 desaturase. Identification of important gene transcripts within insect pests permits them to be targeted with RNA interference, RNAi, strategies. The glassy-winged sharpshooter, GWSS (Hemiptera: Cicadellidae; Homalodis...

  13. A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone.

    PubMed

    Serra, Montserrat; Piña, Benjamin; Abad, José Luis; Camps, Francisco; Fabriàs, Gemma

    2007-10-16

    The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Delta(13) desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Delta/ole1Delta yeast mutants (both elongase 1 and Delta(9) desaturase-deficient) for functional assays. The construct fully rescued the Deltaole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Delta(11) desaturase, Delta(11) acetylenase, and Delta(13) desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. PMID:17921252

  14. A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone

    PubMed Central

    Serra, Montserrat; Piña, Benjamin; Abad, José Luis; Camps, Francisco; Fabriàs, Gemma

    2007-01-01

    The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Δ13 desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Δ/ole1Δ yeast mutants (both elongase 1 and Δ9 desaturase-deficient) for functional assays. The construct fully rescued the Δole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Δ11 desaturase, Δ11 acetylenase, and Δ13 desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. PMID:17921252

  15. The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

    PubMed

    Kuah, Meng-Kiat; Jaya-Ram, Annette; Shu-Chien, Alexander Chong

    2015-03-01

    The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores. PMID:25542509

  16. Δ6-fatty acid desaturase and fatty acid elongase mRNA expression, phagocytic activity and weight-to-length relationships in channel catfish (Ictalurus punctatus) fed alternative diets with soy oil and a probiotic.

    PubMed

    Santerre, A; Téllez-Bañuelos, M C; Casas-Solís, J; Castro-Félix, P; Huízar-López, M R; Zaitseva, G P; Horta-Fernández, J L; Trujillo-García, E A; de la Mora-Sherer, D; Palafox-Luna, J A; Juárez-Carrillo, E

    2015-01-01

    A time-course feeding trial was conducted for 120 days on juvenile channel catfish (Ictalurus punctatus) to study the effects of diets differing in oil source (fish oil or soy oil) and supplementation with a commercial probiotic. Relative levels of Δ6-fatty acid desaturase (Δ6-FAD) and fatty acid elongase (FAE) expression were assessed in brain and liver tissues. Both genes showed similar expression levels in all groups studied. Fish weight-to-length relationships were evaluated using polynomial regression analyses, which identified a burst in weight and length in the channel catfish on day 105 of treatment; this increase was related to an increase in gene expression. Mid-intestinal lactic acid bacterium (LAB) count was determined according to morphological and biochemical criteria using API strips. There was no indication that intestinal LAB count was affected by the modified diets. The Cunningham glass adherence method was applied to evaluate phagocytic cell activity in peripheral blood. Reactive oxygen species (ROS) generation was assessed through the respiratory burst activity of spleen macrophages by the NBT reduction test. Probiotic-supplemented diets provided a good substrate for innate immune system function; the phagocytic index was significantly enhanced in fish fed soy oil and the probiotic, and at the end of the experimental period, ROS production increased in fish fed soy oil. The substitution of fish oil by soy oil is recommended for food formulation and will contribute to promoting sustainable aquaculture. Probiotics are also recommended for channel catfish farming as they may act as immunonutrients. PMID:26400353

  17. Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

    PubMed Central

    Merlo, A O; Cowen, N; Delate, T; Edington, B; Folkerts, O; Hopkins, N; Lemeiux, C; Skokut, T; Smith, K; Woosley, A; Yang, Y; Young, S; Zwick, M

    1998-01-01

    Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize. PMID:9761789

  18. Oleic acid synthesized by stearoyl-CoA desaturase (SCD-1) in the lateral periventricular zone of the developing rat brain mediates neuronal growth, migration and the arrangement of prospective synapses.

    PubMed

    Polo-Hernández, Erica; Tello, Vega; Arroyo, Angel A; Domínguez-Prieto, Marta; de Castro, Fernando; Tabernero, Arantxa; Medina, José M

    2014-06-27

    Our previous work has shown that oleic acid synthesized by astrocytes in response to serum albumin behaves as a neurotrophic factor in neurons, upregulating the expression of GAP-43 and MAP-2 proteins, which are respectively markers of axonal and dendrite growth. In addition, oleic acid promoted neuron migration and aggregation, resulting in clusters of neurons connected each other by the newly formed neurites. In this work we show that the presence of albumin or albumin plus oleic acid increases neuron migration in cultured explants of the lateral periventricular zone, resulting in an increase in the number of GAP-43-positive neurons leaving the explant. Upon silencing stearoyl-CoA desaturase-1 (SCD-1), a key enzyme in oleic acid synthesis by RNA of interference mostly prevented the effect of albumin but not that of albumin plus oleic acid, suggesting that the oleic acid synthesized due to the effect of albumin would be responsible for the increase in neuron migration. Oleic acid increased doublecortin (DCX) expression in cultured neurons, explants and organotypic slices, suggesting that DCX may mediate in the effect of oleic acid on neuron migration. The effect of oleic acid on neuron migration may be destined for the formation of synapses because the presence of oleic acid increased the expression of synaptotagmin and that of postsynaptic density protein (PDS-95), respectively markers of the pre- and postsynaptic compartments. In addition, confocal microscopy revealed the occurrence of points of colocalization between synaptotagmin and PDS-95, which is consistent with the idea that oleic acid promotes synapse arrangement. PMID:24836198

  19. Cell membrane fatty acid changes and desaturase expression of Saccharomyces bayanus exposed to high pressure homogenization in relation to the supplementation of exogenous unsaturated fatty acids

    PubMed Central

    Serrazanetti, Diana I.; Patrignani, Francesca; Russo, Alessandra; Vannini, Lucia; Siroli, Lorenzo; Gardini, Fausto; Lanciotti, Rosalba

    2015-01-01

    Aims: The aim of this work was to study the responses of Saccharomyces bayanus cells exposed to sub-lethal high-pressure homogenization (HPH) and determine whether the plasmatic membrane can sense HPH in the presence, or absence, of exogenous unsaturated fatty acids (UFAs) in the growth medium. Methods and Results: High-pressure homogenization damaged and caused the collapse of cell walls and membranes of a portion of cells; however, HPH did not significantly affect S. bayanus cell viability (less than 0.3 Log CFU ml-1). HPH strongly affected the membrane fatty acid (FA) composition by increasing the percentage of total UFA when compared with saturated fatty acids. The gene expression showed that the transcription of OLE1, ERG3, and ERG11 increased after HPH. The presence of exogenous UFA abolished HPH-induced effects on the OLE1 and ERG3 genes, increased the percentage of membrane lipids and decreased the expression of OLE1 and ERG3 within 30 min of treatment. Conclusion: The results suggest a key role for UFA in the microbial cell response to sub-lethal stress. In addition, these data provide insight into the molecular basis of the response of S. bayanus to this innovative technology. Significance and Impact of the Study: Elucidation of the mechanism of action for sub-lethal HPH will enable the utilization of this technology to modulate the starter performance at the industrial scale. PMID:26528258

  20. Identification of Primula "front-end" desaturases with distinct n-6 or n-3 substrate preferences.

    PubMed

    Sayanova, Olga; Haslam, Richard; Venegas-Calerón, Mónica; Napier, Johnathan A

    2006-11-01

    cDNA clones encoding cytochrome b(5) fusion desaturases were isolated from Primula cortusoides L. and Primula luteola Ruprecht, species previously shown to preferentially accumulate either n-6 or n-3 Delta6-desaturated fatty acids, respectively. Functional characterisation of these desaturases in yeast revealed that the recombinant Primula enzymes displayed substrate preferences, resulting in the predominant synthesis of either gamma-linolenic acid (n-6) or stearidonic acid (n-3). Independent expression of the two Primula desaturases in transgenic Arabidopsis thaliana confirmed these results, with gamma-linolenic acid and stearidonic acid accumulating in both leaf and seed tissues to different levels, depending on the substrate specificity of the desaturase. Targeted lipid analysis of transgenic Arabidopsis lines revealed the presence of Delta6-desaturated fatty acids in the acyl-CoA pools of leaf but not seed tissue. The implications for the transgenic synthesis of C(20) polyunsaturated fatty acids via the elongation of Delta6-desaturated fatty acids are discussed, as is the potential of using Primula desaturases in the synthesis of C(18) n-3 polyunsaturated fatty acids such as stearidonic acid. PMID:16773377

  1. Lipid redistribution by α-linolenic acid-rich chia seed inhibits stearoyl-CoA desaturase-1 and induces cardiac and hepatic protection in diet-induced obese rats.

    PubMed

    Poudyal, Hemant; Panchal, Sunil K; Waanders, Jennifer; Ward, Leigh; Brown, Lindsay

    2012-02-01

    Chia seeds contain the essential fatty acid, α-linolenic acid (ALA). This study has assessed whether chia seeds attenuated the metabolic, cardiovascular and hepatic signs of a high-carbohydrate, high-fat (H) diet [carbohydrates, 52% (wt/wt); fat, 24% (wt/wt) with 25% (wt/vol) fructose in drinking water] in rats. Diets of the treatment groups were supplemented with 5% chia seeds after 8 weeks on H diet for a further 8 weeks. Compared with the H rats, chia seed-supplemented rats had improved insulin sensitivity and glucose tolerance, reduced visceral adiposity, decreased hepatic steatosis and reduced cardiac and hepatic inflammation and fibrosis without changes in plasma lipids or blood pressure. Chia seeds induced lipid redistribution with lipid trafficking away from the visceral fat and liver with an increased accumulation in the heart. The stearoyl-CoA desaturase-1 products were depleted in the heart, liver and the adipose tissue of chia seed-supplemented rats together with an increase in the substrate concentrations. The C18:1trans-7 was preferentially stored in the adipose tissue; the relatively inert C18:1n-9 was stored in sensitive organs such as liver and heart and C18:2n-6, the parent fatty acid of the n-6 pathway, was preferentially metabolized. Thus, chia seeds as a source of ALA induce lipid redistribution associated with cardioprotection and hepatoprotection. PMID:21429727

  2. The delta 6 desaturase knock out mouse reveals that immunomodulatory effects of essential n-6 and n-3 polyunsaturated fatty acids are both independent of and dependent upon conversion.

    PubMed

    Monk, Jennifer M; Liddle, Danyelle M; Cohen, Daniel J A; Tsang, Denis H; Hillyer, Lyn M; Abdelmagid, Salma A; Nakamura, Manabu T; Power, Krista A; Ma, David W L; Robinson, Lindsay E

    2016-06-01

    Typically fatty acids (FA) exert differential immunomodulatory effects with n-3 [α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and n-6 [linoleic acid (LA) and arachidonic acid (AA)] exerting anti- and pro-inflammatory effects, respectively. This over-simplified interpretation is confounded by a failure to account for conversion of the parent FA (LA and ALA) to longer-chain bioactive products (AA and EPA/DHA, respectively), thereby precluding discernment of the immunomodulatory potential of specific FA. Therefore, we utilized the Δ6-desaturase model, wherein knockout mice (D6KO) lack the Fads2 gene encoding for the rate-limiting enzyme that initiates FA metabolism, thereby providing a model to determine specific FA immunomodulatory effects. Wild-type (WT) and D6KO mice were fed one of four isocaloric diets differing in FA source (9weeks): corn oil (LA-enriched), arachidonic acid single cell oil (AA-enriched), flaxseed oil (ALA-enriched) or menhaden fish oil (EPA/DHA-enriched). Splenic mononuclear cell cytokine production in response to lipopolysaccharide (LPS), T-cell receptor (TCR) and anti-CD40 stimulation was determined. Following LPS stimulation, AA was more bioactive compared to LA, by increasing inflammatory cytokine production of IL-6 (1.2-fold) and TNFα (1.3-fold). Further, LPS-stimulated IFNγ production in LA-fed D6KO mice was reduced 5-fold compared to LA-fed WT mice, indicating that conversion of LA to AA was necessary for cytokine production. Conversely, ALA exerted an independent immunomodulatory effect from EPA/DHA and all n-3 FA increased LPS-stimulated IL-10 production versus LA and AA. These data definitively identify specific immunomodulatory effects of individual FA and challenge the simplified view of the immunomodulatory effects of n-3 and n-6 FA. PMID:27142734

  3. Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity.

    PubMed

    Matousková, Petra; Pichová, Iva; Svatos, Ales

    2007-06-01

    The pheromone blend produced by the tobacco hornworm moth (Manduca sexta) (L.) female is unusually complex and contains two conjugated dienals and trienals together with two monounsaturated alkenals. Here, we describe the identification and construction of two genes encoding MsexKPSE and MsexAPTQ desaturases from a cDNA library prepared from the total RNA of the M. sexta pheromone gland. The MsexKPSE desaturase shares a high degree of similarity with Delta(9)-desaturases from different moth species. The functional expression of MsexAPTQ desaturase in Saccharomyces cerevisiae followed by a detailed GC-MS analysis of fatty acid methyl esters (FAME) and their derivatized products and gas-phase Fourier transform infrared (FTIR) spectroscopy of the extracted FAME confirms that this enzyme is a bifunctional Z-Delta(11)-desaturase. MsexAPTQ desaturase catalyses the production of Z11-hexadecenoate (Z11-16) and Z10E12- and E10E12-hexadecadienoates (Z10E12-16) via 1,4-desaturation of the Z11-16 substrate. The stereochemistry of 1,4-desaturation and formation of isomers is discussed. PMID:17517337

  4. Corn oil supplementation to steers grazing endophyte-free tall fescue. II. Effects on longissimus muscle and subcutaneous adipose fatty acid composition and stearoyl-CoA desaturase activity and expression.

    PubMed

    Pavan, E; Duckett, S K

    2007-07-01

    Eighteen steers were used to evaluate the effect of supplemental corn oil level to steers grazing endophyte-free tall fescue on fatty acid composition of LM, stearoyl CoA desaturase (SCD) activity and expression as well as cellularity in s.c. adipose. Corn oil was supplemented (g/kg of BW) at 0 (none), 0.75 (medium), and 1.5 (high). Cottonseed hulls were used as a carrier for the corn oil and were supplemented according to pasture availability (0.7 to 1% of BW). Steers were finished on a rotationally grazed, tall fescue pasture for 116 d. Fatty acid composition of LM, s.c. adipose, and diet was determined by GLC. Total linoleic acid intake increased linearly (P < 0.01) with corn oil supplementation (90.7, 265.1, and 406.7 g in none, medium, and high, respectively). Oil supplementation linearly reduced (P < 0.05) myristic, palmitic, and linolenic acid percentage in LM and s.c. adipose. Vaccenic acid (C18:1 t11; VA) percentage was 46 and 32% greater (linear, P = 0.02; quadratic, P = 0.01) for medium and high, respectively, than none, regardless of tissue. Effect of oil supplementation on CLA cis-9, trans-11 was affected by type of adipose tissue (P < 0.01). In the LM, CLA cis-9, trans-11 isomer was 25% greater for medium than for none and intermediate for high, whereas CLA cis-9, trans-11 CLA isomer was 48 and 33% greater in s.c. adipose tissue for medium and high than for none, respectively. Corn oil linearly increased (P acid and CLA trans-10, cis-12; however, values were low (<0.35 and <0.035% of total fatty acids, respectively). Oil supplementation did not change (P > 0.05) the percentage of total SFA, MUFA, or PUFA but linearly increased (P = 0.03) n-6:n-3 ratio from 2.4 to 2.9 in none and high, respectively. Among tissues, total SFA and MUFA were greater in s.c. adipose than LM, whereas total PUFA, n-6, and n-3 fatty acids and the n-6:n-3 ratio were lower. Trans-10 octadecenoic acid, VA, and CLA trans-10, cis-12 were greater (P

  5. Echium oil increased the expression of a Δ4 Fads2 fatty acyl desaturase and the deposition of n-3 long-chain polyunsaturated fatty acid in comparison with linseed oil in striped snakehead (Channa striata) muscle.

    PubMed

    Jaya-Ram, Annette; Shu-Chien, Alexander Chong; Kuah, Meng-Kiat

    2016-08-01

    Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species. PMID:26842427

  6. Genetic variation in lipid desaturases and its impact on the development of human disease

    PubMed Central

    2010-01-01

    Perturbations in lipid metabolism characterize many of the chronic diseases currently plaguing our society, such as obesity, diabetes, and cardiovascular disease. Thus interventions that target plasma lipid levels remain a primary goal to manage these diseases. The determinants of plasma lipid levels are multi-factorial, consisting of both genetic and lifestyle components. Recent evidence indicates that fatty acid desaturases have an important role in defining plasma and tissue lipid profiles. This review will highlight the current state-of-knowledge regarding three desaturases (Scd-1, Fads1 and Fads2) and their potential roles in disease onset and development. Although research in rodent models has provided invaluable insight into the regulation and functions of these desaturases, the extent to which murine research can be translated to humans remains unclear. Evidence emerging from human-based research demonstrates that genetic variation in human desaturase genes affects enzyme activity and, consequently, disease risk factors. Moreover, this genetic variation may have a trans-generational effect via breastfeeding. Therefore inter-individual variation in desaturase function is attributed to both genetic and lifestyle components. As such, population-based research regarding the role of desaturases on disease risk is challenged by this complex gene-lifestyle paradigm. Unravelling the contribution of each component is paramount for understanding the inter-individual variation that exists in plasma lipid profiles, and will provide crucial information to develop personalized strategies to improve health management. PMID:20565855

  7. Knockdown of delta-5-desaturase promotes the anti-cancer activity of dihomo-γ-linolenic acid and enhances the efficacy of chemotherapy in colon cancer cells expressing COX-2.

    PubMed

    Xu, Yi; Yang, Xiaoyu; Zhao, Pinjing; Yang, Zhongyu; Yan, Changhui; Guo, Bin; Qian, Steven Y

    2016-07-01

    Cyclooxygenase (COX), commonly overexpressed in cancer cells, is a major lipid peroxidizing enzyme that metabolizes polyunsaturated fatty acids (ω-3s and ω-6s). The COX-catalyzed free radical peroxidation of arachidonic acid (ω-6) can produce deleterious metabolites (e.g. 2-series prostaglandins) that are implicated in cancer development. Thus, COX inhibition has been intensively investigated as a complementary therapeutic strategy for cancer. However, our previous study has demonstrated that a free radical-derived byproduct (8-hydroxyoctanoic acid) formed from COX-catalyzed peroxidation of dihomo-γ-linolenic acid (DGLA, the precursor of arachidonic acid) can inhibit colon cancer cell growth. We thus hypothesize that the commonly overexpressed COX in cancer (~90% of colon cancer patients) can be taken advantage to suppress cell growth by knocking down delta-5-desaturase (D5D, a key enzyme that converts DGLA to arachidonic acid). In addition, D5D knockdown along with DGLA supplement may enhance the efficacy of chemotherapeutic drugs. After knocking down D5D in HCA-7 colony 29 cells and HT-29 cells (human colon cancer cell lines with high and low COX levels, respectively), the antitumor activity of DGLA was significantly enhanced along with the formation of a threshold range (~0.5-1.0μM) of 8-hydroxyoctanoic acid. In contrast, DGLA treatment did not inhibit cell growth when D5D was not knocked down and only limited amount of 8-hydroxyoctanoic acid was formed. D5D knockdown along with DGLA treatment also enhanced the cytotoxicities of various chemotherapeutic drugs, including 5-fluorouracil, regorafenib, and irinotecan, potentially through the activation of pro-apoptotic proteins, e.g. p53 and caspase 9. For the first time, we have demonstrated that the overexpressed COX in cancer cells can be utilized in suppressing cancer cell growth. This finding may provide a new option besides COX inhibition to optimize cancer therapy. The outcome of this translational

  8. Knockdown of delta-5-desaturase promotes the anti-cancer activity of dihomo-γ-linolenic acid and enhances the efficacy of chemotherapy in colon cancer cells expressing COX-2

    PubMed Central

    Xu, Yi; Yang, Xiaoyu; Zhao, Pinjing; Yang, Zhongyu; Yan, Changhui; Guo, Bin; Qian, Steven Y.

    2016-01-01

    Cyclooxygenase (COX), commonly overexpressed in cancer cells, is a major lipid peroxidizing enzyme that metabolizes polyunsaturated fatty acids (ω-3s and ω-6s). The COX-catalyzed free radical peroxidation of arachidonic acid (ω-6) can produce deleterious metabolites (e.g. 2-series prostaglandins) that are implicated in cancer development. Thus, COX inhibition has been intensively investigated as a complementary therapeutic strategy for cancer. However, our previous study has demonstrated that a free radical-derived by product (8-hydroxyoctanoic acid) formed from COX-catalyzed peroxidation of dihomo-γ-linolenic acid (DGLA, the precursor of arachidonic acid) can inhibit colon cancer cell growth. We thus hypothesize that the commonly overexpressed COX in cancer (~90% of colon cancer patients) can be taken advantage to suppress cell growth by knocking down delta-5-desaturase (D5D, a key enzyme that converts DGLA to arachidonic acid). In addition, D5D knockdown along with DGLA supplement may enhance the efficacy of chemotherapeutic drugs. After knocking down D5D in HCA-7 colony 29 cells and HT-29 cells (human colon cancer cell lines with high and low COX levels, respectively), the antitumor activity of DGLA was significantly enhanced along with the formation of a threshold range (~0.5–1.0 µM) of 8-hydroxyoctanoic acid. In contrast, DGLA treatment did not inhibit cell growth when D5D was not knocked down and only limited amount of 8-hydroxyoctanoic acid was formed. D5D knockdown along with DGLA treatment also enhanced the cytotoxicities of various chemotherapeutic drugs, including 5-fluorouracil, regorafenib, and irinotecan, potentially through the activation of pro-apoptotic proteins, e.g. p53 and caspase 9. For the first time, we have demonstrated that the overexpressed COX in cancer cells can be utilized in suppressing cancer cell growth. This finding may provide a new option besides COX inhibition to optimize cancer therapy. The outcome of this translational

  9. Regulation of Tissue LC-PUFA Contents, Δ6 Fatty Acyl Desaturase (FADS2) Gene Expression and the Methylation of the Putative FADS2 Gene Promoter by Different Dietary Fatty Acid Profiles in Japanese Seabass (Lateolabrax japonicus)

    PubMed Central

    Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53±0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter. PMID:24498178

  10. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs.

    PubMed Central

    Shanklin, J; Somerville, C

    1991-01-01

    Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently. However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated. Images PMID:2006187

  11. A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High oleic acid, such as is found in olive oil, is desirable for the healthy cholesterol-lowering benefits. The oxidative stability of the oil with high oleic acid also gives longer “shelve life” for peanut products. These benefits drive the breeding effort toward developing high oleic peanuts worl...

  12. New mutation in Delta-9-Stearoyl-Acyl Carrier Protein desaturase gene associated with enhanced stearic acid levels in soybean seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean [Glycine max (L.) Merr] oil from conventional cultivars typically contains ~3% stearic acid of the total seed oil. Increased stearic acid concentration in the seed oil of soybeans is desirable from both food and industrial use stand-points. To date a small number of mutants have been develop...

  13. Development of markers for Delta9-Stearoyl-ACP-Desaturase (SAD) to screen for cold acclimation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delta 9-Stearoyl-acyl carrier protein (ACP) desaturase (SAD) is an important enzyme of fatty acid biosynthesis in higher plants. Located in the plastid stroma, SAD catalyzes the desaturation of stearoyl-ACP to oleyl-ACP. SAD plays a key role in determining the ratio of saturated fatty acids to unsat...

  14. Liver X receptor α promotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1 in an SREBP-1-dependent manner.

    PubMed

    Yao, D W; Luo, J; He, Q Y; Xu, H F; Li, J; Shi, H B; Wang, H; Chen, Z; Loor, J J

    2016-08-01

    Stearoyl-coenzyme A desaturase 1 (SCD1) is a pivotal enzyme in the biosynthesis of monounsaturated fatty acids (MUFA). It is tightly regulated by transcription factors that control lipogenesis. In nonruminants, liver X receptor α (LXRα) is a nuclear receptor and transcription factor that acts as a key sensor of cholesterol and lipid homeostasis. However, the mechanism whereby LXRα regulates the expression and transcriptional activity of SCD1 in ruminant mammary cells remains unknown. In this study with goat mammary epithelial cells (GMEC), the LXRα agonist T 4506585 (T09) markedly enhanced the mRNA expression of SCD1 and sterol regulatory element binding factor 1 (SREBF1). The concentrations of C16:1 and C18:1 and their desaturation indices also were increased by LXRα activation. However, knockdown of LXRα did not alter the mRNA expression of SCD1. Although SCD1 was repressed by SREBF1 knockdown, T09 significantly increased SCD1 expression. Further analysis revealed that the SCD1 promoter activity was activated by LXRα overexpression. The goat SCD1 promoter contains 2 LXR response elements (LXRE), 1 sterol response element (SRE), and 1 nuclear factor Y (NF-Y) binding site. Site-directed mutagenesis of LXRE1, LXRE2, or SRE alone did not eliminate the upregulation of SCD1 when LXRα was overexpressed. In contrast, when NF-Y alone or in combination with SRE was mutated simultaneously, the basal transcriptional activity of the SCD1 promoter was markedly decreased and did not respond to LXRα overexpression. Furthermore, when SREBF1 was knocked down, overexpression of LXRα did not affect the promoter activity of SCD1. Together, these data suggest that LXRα regulates the expression of SCD1 through increasing SREBP-1 abundance to promote interaction with SRE and NF-Y binding sites. The present study provides evidence that LXRα is involved in the synthesis of MUFA in the goat mammary gland through an indirect mechanism. PMID:27209141

  15. Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

    PubMed Central

    Mustardy, L; Los, D A; Gombos, Z; Murata, N

    1996-01-01

    There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes. Images Fig. 1 Fig. 2 Fig. 3 PMID:11607709

  16. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared towards the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically-expressed enzymes. Here we show that the addit...

  17. Evidence that the Yeast Desaturase Ole1p Exists as a Dimer In Vivo

    SciTech Connect

    Lou, Y.; Shanklin, J.

    2010-06-18

    Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the {Delta}{sup o}-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an {approx}75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.

  18. Evolution of the integral membrane desaturase gene family in moths and flies.

    PubMed Central

    Knipple, Douglas C; Rosenfield, Claire-Lise; Nielsen, Rasmus; You, Kyung Man; Jeong, Seong Eun

    2002-01-01

    Lepidopteran insects use sex pheromones derived from fatty acids in their species-specific mate recognition system. Desaturases play a particularly prominent role in the generation of structural diversity in lepidopteran pheromone biosynthesis as a result of the diverse enzymatic properties they have evolved. These enzymes are homologous to the integral membrane desaturases, which play a primary role in cold adaptation in eukaryotic cells. In this investigation, we screened for desaturase-encoding sequences in pheromone glands of adult females of eight lepidopteran species. We found, on average, six unique desaturase-encoding sequences in moth pheromone glands, the same number as is found in the genome database of the fly, Drosophila melanogaster, vs. only one to three in other characterized eukaryotic genomes. The latter observation suggests the expansion of this gene family in insects before the divergence of lepidopteran and dipteran lineages. We present the inferred homology relationships among these sequences, analyze nonsynonymous and synonymous substitution rates for evidence of positive selection, identify sequence and structural correlates of three lineages containing characterized enzymatically distinct desaturases, and discuss the evolution of this sequence family in insects. PMID:12524345

  19. Factors affecting the palmitoyl-coenzyme A desaturase of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Volkmann, C. M.

    1975-01-01

    The activity and stability of the palmitoyl-coenzyme A (CoA) desaturase complex of Saccharomyces cerevisiae was influenced by several factors. Cells, grown nonaerobically and then incubated with glucose, either in air or under N2, showed a marked increase in desaturase activity. Cycloheximide, added during such incubations, prevented the increase in activity, suggesting de novo synthesis. The stability of the desaturase from cells grown nonaerobically was affected by subsequent treatment of the cells; enzyme from freshly harvested cells, or from cells that were then shaken under nitrogen, readily lost activity upon washing or during density gradient analysis, whereas aerated cells, in the presence or absence of glucose, yielded stable enzyme preparations. The loss of activity in nonaerobic preparations could be reversed by adding soluble supernatant from these homogenates and could be prevented by growing the cells in the presence of palmitoleic acid and ergosterol, but not with several other lipids tested.

  20. Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

    NASA Astrophysics Data System (ADS)

    Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Pan, Kehou; Pan, Jin; Yang, Guanpin

    2011-03-01

    A gene ( NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae). The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids (LCPUFAs), including eicosapentaenoic acid (20:5n-3, EPA). The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NANOC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid (LA) and α-linolenic acid (ALA).

  1. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    SciTech Connect

    Van De Loo, F.J.; Broun, P.; Turner, S.; Somerville, C.

    1995-07-18

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.

  2. Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

    SciTech Connect

    Alloatti, Andres; Gupta, Shreedhara; Gualdron-Lopez, Melisa; Nguewa, Paul A.; Altabe, Silvia G.; Deumer, Gladys; Wallemacq, Pierre; Michels, Paul A.M.; Uttaro, Antonio D.

    2011-08-26

    Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

  3. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

    PubMed Central

    Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-01-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  4. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

    PubMed

    Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-10-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  5. Identification and Functional Analysis of Delta-9 Desaturase, a Key Enzyme in PUFA Synthesis, Isolated from the Oleaginous Diatom Fistulifera

    PubMed Central

    Muto, Masaki; Kubota, Chihiro; Tanaka, Masayoshi; Satoh, Akira; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

    2013-01-01

    Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ9 desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ9 acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ9 desaturase. Furthermore, the function of these Δ9 desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ9 acyl-CoA desaturases showed Δ9 desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ9 desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain. PMID:24039966

  6. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs

    SciTech Connect

    Shanklin, J.; Somerville, C. )

    1991-03-15

    Stearoyl-acyl-carrier-protein (ACP) desaturase was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the {Delta}{sup 9} desaturase is developmentally regulated.

  7. Identification and functional analysis of the genes encoding Delta6-desaturase from Ribes nigrum.

    PubMed

    Song, Li-Ying; Lu, Wan-Xiang; Hu, Jun; Zhang, Yan; Yin, Wei-Bo; Chen, Yu-Hong; Hao, Shan-Ting; Wang, Bai-Lin; Wang, Richard R-C; Hu, Zan-Min

    2010-06-01

    Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity. PMID:20231328

  8. A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans

    PubMed Central

    2012-01-01

    Background Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Δ9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. Results Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Δ9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Δ9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. Conclusions The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Δ9 desaturases places C.elegans into a catabolic state compared with wildtype controls. PMID:22264337

  9. Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.

    PubMed

    Sperling, P; Libisch, B; Zähringer, U; Napier, J A; Heinz, E

    2001-04-15

    The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves. PMID:11368168

  10. Characterization of soluble acyl-ACP desaturases from Camelina sativa, Macadamia tetraphylla and Dolichandra unguis-cati.

    PubMed

    Rodríguez, Manuel Fernando Rodríguez; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-04-15

    Acyl-acyl carrier protein (ACP) desaturases (EC 1.14.19.2) are soluble enzymes that catalyse the insertion of a double bond into saturated fatty acid bound in saturated acyl chains bound to ACP in higher plants, producing cis-monounsaturated fatty acids. Three types of soluble acyl-ACP desaturases have been described: Δ(9)-acyl-ACP, Δ(6)-acyl-ACP and Δ(4)-acyl-ACP desaturases, which differ in the substrate specificity and the position in which the double bond is introduced. In the present work, Camelina sativa (CsSAD), Macadamia tetraphylla (MtSAD) and Dolichandra unguis-cati (DuSAD) desaturases were cloned, sequenced and characterized. Single copies of CsSAD, MtSAD and DuSAD with three, one and two different alleles, respectively, were found. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization in protein extracts. The recombinant CsSAD enzyme showed 300-fold higher specificity towards 18:0-ACP than 16:0-ACP. Similar profile exhibited MtSAD although the differences in the specificity were lower, around 170-fold higher for 18:0-ACP than 16:0-ACP. Furthermore, DuSAD presented a profile showing preference towards 16:0-ACP against 18:0-ACP, around twice more, being so a Δ(9) palmitoyl-ACP desaturase. Also, we reported the expression profile of CsSAD, which showed the highest levels of expression in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm. Moreover, the possibility to express a new desaturase in C. sativa (oilseed crop that store high levels of oil and is easy to transform) to create a new line rich in short monounsaturated fatty acid is discussed. PMID:25765361

  11. Stabilization of the yeast desaturase system by low levels of oxygen

    NASA Technical Reports Server (NTRS)

    Volkmann, C. M.; Klein, H. P.

    1983-01-01

    The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

  12. Bioengineering resistance to phytoene desaturase inhibitors in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

  13. Effects of FADS and ELOVL polymorphisms on indexes of desaturase and elongase activities: results from a pre-post fish oil supplementation.

    PubMed

    Cormier, Hubert; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Julien, Pierre; Vohl, Marie-Claude

    2014-11-01

    Polymorphisms (SNPs) within the FADS gene cluster and the ELOVL gene family are believed to influence enzyme activities after an omega-3 (n-3) fatty acid (FA) supplementation. The objectives of the study are to test whether an n-3 supplementation is associated with indexes of desaturase and elongase activities in addition to verify whether SNPs in the FADS gene cluster and the ELOVL gene family modulate enzyme activities of desaturases and elongases. A total 208 subjects completed a 6-week supplementation period with 5 g/day of fish oil (1.9-2.2 g/day of EPA + 1.1 g/day of DHA). FA profiles of plasma phospholipids were obtained by gas chromatography (n = 210). Desaturase and elongase indexes were estimated using product-to-precursor ratios. Twenty-eight SNPs from FADS1, FADS2, FADS3, ELOVL2 and ELOVL5 were genotyped using TaqMan technology. Desaturase indexes were significantly different after the 6-week n-3 supplementation. The index of δ-5 desaturase activity increased by 25.7 ± 28.8 % (p < 0.0001), whereas the index of δ-6 desaturase activity decreased by 17.7 ± 18.2 % (p < 0.0001) post-supplementation. Index of elongase activity decreased by 39.5 ± 27.9 % (p < 0.0001). Some gene-diet interactions potentially modulating the enzyme activities of desaturases and elongases involved in the FA metabolism post-supplementation were found. SNPs within the FADS gene cluster and the ELOVL gene family may play an important role in the enzyme activity of desaturases and elongases, suggesting that an n-3 FAs supplementation may affect PUFA metabolism. PMID:25367143

  14. Non-redundant Contribution of the Plastidial FAD8 ω-3 Desaturase to Glycerolipid Unsaturation at Different Temperatures in Arabidopsis.

    PubMed

    Román, Ángela; Hernández, María L; Soria-García, Ángel; López-Gomollón, Sara; Lagunas, Beatriz; Picorel, Rafael; Martínez-Rivas, José Manuel; Alfonso, Miguel

    2015-11-01

    Plastidial ω-3 desaturase FAD7 is a major contributor to trienoic fatty acid biosynthesis in the leaves of Arabidopsis plants. However, the precise contribution of the other plastidial ω-3 desaturase, FAD8, is poorly understood. Fatty acid and lipid analysis of several ω-3 desaturase mutants, including two insertion lines of AtFAD7 and AtFAD8, showed that FAD8 partially compensated the disruption of the AtFAD7 gene at 22 °C, indicating that FAD8 was active at this growth temperature, contrasting to previous observations that circumscribed the FAD8 activity at low temperatures. Our data revealed that FAD8 had a higher selectivity for 18:2 acyl-lipid substrates and a higher preference for lipids other than galactolipids, particularly phosphatidylglycerol, at any of the temperatures studied. Differences in the mechanism controlling AtFAD7 and AtFAD8 gene expression at different temperatures were also detected. Confocal microscopy and biochemical analysis of FAD8-YFP over-expressing lines confirmed the chloroplast envelope localization of FAD8. Co-localization experiments suggested that FAD8 and FAD7 might be located in close vicinity in the envelope membrane. FAD8-YFP over-expressing lines showed a specific increase in 18:3 fatty acids at 22 °C. Together, these results indicate that the function of both plastidial ω-3 desaturases is coordinated in a non-redundant manner. PMID:26079601

  15. Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase1[OPEN

    PubMed Central

    Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

    2015-01-01

    Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. PMID:26224800

  16. Relationship between Body Mass Index, C-Peptide, and Delta-5-Desaturase Enzyme Activity Estimates in Adult Males

    PubMed Central

    Pickens, C. Austin; Matsuo, Karen H.; Fenton, Jenifer I.

    2016-01-01

    Obesity, in particular abdominal obesity, alters the composition of plasma and tissue fatty acids (FAs), which contributes to inflammation and insulin resistance. FA metabolism is modulated by desaturases and may affect adipokine and insulin secretion. Therefore, we examined relationships between adipokines, a marker of insulin production, and plasma FA desaturase enzyme activity estimates (EAEs) in obesity. Plasma phospholipid (PPL) FAs were isolated from 126 males (ages 48 to 65 years), derivatized, and analyzed using gas chromatography. Delta-6 desaturase (D6D) and delta-5 desaturase (D5D) EAEs were calculated as the ratio of PPL 20:3/18:2 and 20:4/20:3, respectively. In body mass index (BMI) and waist circumference (WC) adjusted polytomous logistic regression analyses, PPL FAs and FA desaturase EAEs were associated with C-peptide and adiponectin. Individuals with elevated D6D EAEs were less likely (OR 0.33) to have serum adiponectin concentrations > 5.37 μg/mL, compared with adiponectin concentrations ≤ 3.62 μg/mL. Individuals with increased D5D EAEs were less likely (OR 0.8) to have C-peptide concentrations ≥ 3.32 ng/mL, and > 1.80 and ≤ 3.29 ng/mL, compared with those with C-peptide ≤ 1.76 ng/mL. The proinflammatory cytokine tumor necrosis factor-α (TNF- α) was positively associated with C-peptide, but TNF- α was not associated with the D5D EAE. C-peptide and adiponectin concentrations are associated with specific PPL FAs and FA desaturase EAEs. The relationship between C-peptide concentrations and D5D EAEs remained significant after adjusting for BMI, WC, and TNF-α. Thus, future research should investigate whether D5D inhibition may occur through a C-peptide mediated pathway. PMID:27023786

  17. Dietary whole cottonseed depresses lipogenesis but has no effect on stearoyl coenzyme desaturase activity in bovine subcutaneous adipose tissue.

    PubMed

    Page, A M; Sturdivant, C A; Lunt, D K; Smith, S B

    1997-09-01

    The primary objective of this study was to determine the effect of long-term feeding of whole cottonseed (WCS) on lipogenesis and stearoyl-coenzyme A desaturase activity in growing steers. Brangus steers were fed either a control, cornbased diet (n = 11) or 30% WCS (n = 12). The 30% WCS contributed an estimated 6.6% additional lipid to the diet. Steers fed the added WCS had greater live weights (P = 0.04) and kidney, pelvic, and heart fat (P = 0.005). Subcutaneous fat thickness was not different (P = 0.20) between treatment groups, although WCS elicited an increase in the proportion of large diameter subcutaneous adipocytes. The rate of [U-14C]acetate incorporation into fatty acids in subcutaneous adipose tissue was reduced by dietary WCS (171.4 vs 122.1 nmol x 100 mg adipose tissue-1 x 2 hr-1, P = 0.03), indicating that the increased dietary fat depressed de novo lipogenesis. Hepatic desaturase activity was much lower than that of subcutaneous adipose tissue, a feature common to cattle. We anticipated that added WCS also would depress stearoyl-coenzyme A desaturase activity in subcutaneous adipose tissue and liver due to its cyclopropene fatty acid content. Instead, desaturase activity was numerically (although not significantly) greater in liver (P = 0.37) and adipose tissue (P = 0.23). PMID:9417995

  18. Transcript abundance of the pig stearoyl-CoA desaturase gene has no effect on fatty acid composition in muscle and fat tissues, but its polymorphism within the putative microRNA target site is associated with daily body weight gain and feed conversion ratio.

    PubMed

    Bartz, M; Szydlowski, M; Kociucka, B; Salamon, S; Jeleń, H H; Switonski, M

    2013-01-01

    Fatty acid composition in porcine intramuscular fat affects the dietetic value and technological properties of meat. The stearoyl-CoA desaturase (SCD) gene is a strong positional and functional candidate for fatty acid composition. Our sequence analysis in 4 breeds (Duroc, Pietrain, Polish Landrace, and Polish Large White) revealed a novel SNP in the 5'-flanking sequence and 9 novel SNP and 2 novel indels in the 3' untranslated region (UTR). Transcript level of the SCD in subcutaneous fat was significantly greater than in muscle tissue (n=83; P<0.001) and the interbreed comparison revealed a greater transcript level in the fat tissue of Polish Landrace (P<0.01). We found no association between the abundance of the SCD transcript and fatty acid composition in any of the tissues. We performed an association analysis between 4 SNP (c.-353C>T, c.-233T>C, c.*164A>G, and c.*928G>C), 1 indel (c.*2574_2576delGTC), and production traits in Polish Large White (n=185) and synthetic line 990 (n=243). The most pronounced associations were observed for the c.*928G>C polymorphism, which occurs within a predicted target site for 2 microRNA (ssc-miR-185 and ssc-miR-491). In line 990, this polymorphism was significantly associated with daily BW gain (P<0.04 under the general model) and feed conversion ratio (P<0.0004) but not with fatness traits. The same tendency, but not significant, was observed in the Polish Large White breed. When both breeds were analyzed together, these associations were again highly significant (daily BW gain P<0.003; feed conversion ratio P<0.0001). We conclude that c.*928G>C is a promising marker for both porcine traits. PMID:23048140

  19. Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity.

    PubMed

    Abdul Hamid, Noor Khalidah; Carmona-Antoñanzas, Greta; Monroig, Óscar; Tocher, Douglas R; Turchini, Giovanni M; Donald, John A

    2016-01-01

    Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors. PMID:26943160

  20. Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

    PubMed Central

    Abdul Hamid, Noor Khalidah; Carmona-Antoñanzas, Greta; Monroig, Óscar; Tocher, Douglas R.; Turchini, Giovanni M.; Donald, John A.

    2016-01-01

    Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors. PMID:26943160

  1. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

    PubMed

    Rios-Esteves, Jessica; Resh, Marilyn D

    2013-09-26

    Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  2. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins

    PubMed Central

    Rios-Esteves, Jessica; Resh, Marilyn D.

    2013-01-01

    Summary Wnt proteins contain an unusual lipid modification, palmitoleic acid. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate which is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a, and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  3. [Cloning and functional characterization of phytoene desaturase in Andrographis paniculata].

    PubMed

    Shen, Qin-qin; Li, Li-xia; Zhan, Peng-lin; Wang, Qiang

    2015-10-01

    A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway. PMID:26975098

  4. Identification and characterization of Delta12, Delta6, and Delta5 Desaturases from the green microalga Parietochloris incisa.

    PubMed

    Iskandarov, Umidjon; Khozin-Goldberg, Inna; Cohen, Zvi

    2010-06-01

    The freshwater microalga Parietochloris incisa accumulates, under nitrogen starvation, large amounts of triacylglycerols containing approximately 60% of the omega6 very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. Based on sequence homology, we isolated three cDNA sequences from P. incisa, designated PiDesD12, PiDesD6, PiDesD5. The deduced amino acid sequences of the three genes contained three conserved histidine motifs; the front-end desaturases, PiDes6 and PiDes5, contained a fused N-terminal cytochrome b5 domain. By functional characterization in the yeast Saccharomyces cerevisiae, we confirmed that PiDesD6, PiDesD5 cDNA encode membrane bound desaturases with Delta6, and Delta5 activity, respectively. Both PiDes6 and PiDes5 can indiscriminately desaturate both omega6 and omega3 substrates. A phylogenetic analysis showed that the three genes were homologous to the corresponding desaturases from green microalgae and lower plants that were functionally characterized. Quantitative real-time PCR revealed the concerted expression pattern of all three genes in P. incisa cells subjected to nitrogen starvation, featuring maximum expression level on day 3 of starvation, corresponding to the sharpest increase in the share of arachidonic acid. PMID:20467827

  5. Vertebrate fatty acyl desaturase with Δ4 activity

    PubMed Central

    Li, Yuanyou; Monroig, Oscar; Zhang, Liang; Wang, Shuqi; Zheng, Xiaozhong; Dick, James R.; You, Cuihong; Tocher, Douglas R.

    2010-01-01

    Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n - 6 to produce 18∶3n - 6 that is elongated to 20∶3n - 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above. PMID:20826444

  6. Molecular breeding for introgression of fatty acid desaturase mutant alleles (ahFAD2A and ahFAD2B) enhances oil quality in high and low oil containing peanut genotypes.

    PubMed

    Janila, Pasupuleti; Pandey, Manish K; Shasidhar, Yaduru; Variath, Murali T; Sriswathi, Manda; Khera, Pawan; Manohar, Surendra S; Nagesh, Patne; Vishwakarma, Manish K; Mishra, Gyan P; Radhakrishnan, T; Manivannan, N; Dobariya, K L; Vasanthi, R P; Varshney, Rajeev K

    2016-01-01

    High oleate peanuts have two marketable benefits, health benefits to consumers and extended shelf life of peanut products. Two mutant alleles present on linkage group a09 (ahFAD2A) and b09 (ahFAD2B) control composition of three major fatty acids, oleic, linoleic and palmitic acids which together determine peanut oil quality. In conventional breeding, selection for fatty acid composition is delayed to advanced generations. However by using DNA markers, breeders can reject large number of plants in early generations and therefore can optimize time and resources. Here, two approaches of molecular breeding namely marker-assisted backcrossing (MABC) and marker-assisted selection (MAS) were employed to transfer two FAD2 mutant alleles from SunOleic 95R into the genetic background of ICGV 06110, ICGV 06142 and ICGV 06420. In summary, 82 MABC and 387 MAS derived introgression lines (ILs) were developed using DNA markers with elevated oleic acid varying from 62 to 83%. Oleic acid increased by 0.5-1.1 folds, with concomitant reduction of linoleic acid by 0.4-1.0 folds and palmitic acid by 0.1-0.6 folds among ILs compared to recurrent parents. Finally, high oleate ILs, 27 with high oil (53-58%), and 28 ILs with low oil content (42-50%) were selected that may be released for cultivation upon further evaluation. PMID:26566838

  7. Characterization and regulation of the bovine stearoyl-CoA desaturase gene promoter

    SciTech Connect

    Keating, Aileen F.; Kennelly, John J.; Zhao Fengqi . E-mail: fzhao@uvm.edu

    2006-05-26

    The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36 bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid.

  8. Δ10(E)-Sphingolipid Desaturase Involved in Fusaruside Mycosynthesis and Stress Adaptation in Fusarium graminearum

    PubMed Central

    Tian, Yuan; Zhao, Guo Y.; Fang, Wei; Xu, Qiang; Tan, Ren X.

    2015-01-01

    Sphingolipids are biologically important and structurally distinct cell membrane components. Fusaruside (1) is a 10,11-unsaturated immunosuppressive fungal sphingolipid with medical potentials for treating liver injury and colitis, but its poor natural abundance bottlenecks its druggability. Here, fusaruside is clarified biosynthetically, and its efficacy-related 10,11-double bond can be generated under the regioselective catalysis of an unprecedented Δ10(E)-sphingolipid desaturase (Δ10(E)-SD). Δ10(E)-SD shares 17.7% amino acid sequence similarity with a C9-unmethylated Δ10-sphingolipid desaturase derived from a marine diatom, and 55.7% with Δ8(E)-SD from Fusarium graminearum. Heterologous expression of Δ10(E)-SD in Pichia pastoris has been established to facilitate a reliable generation of 1 through the Δ10(E)-SD catalyzed desaturation of cerebroside B (2), an abundant fungal sphingolipid. Site directed mutageneses show that the conserved histidines of Δ10(E)-SD are essential for the 10,11-desaturation catalysis, which is also preconditioned by the C9-methylation of the substrate. Moreover, Δ10(E)-SD confers improved survival and faster growth to fungal strains at low temperature and high salinity, in parallel with to higher contents of 1 in the mycelia. Collectively, the investigation describes a new Δ10(E)-sphingolipid desaturase with its heterologous expression fundamentalizing a biotechnological supply of 1, and eases the follow-up clarification of the immunosuppression and stress-tolerance mechanism. PMID:25994332

  9. Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

    PubMed

    Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. PMID:25038276

  10. Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua).

    PubMed

    Xue, Xi; Feng, Charles Y; Hixson, Stefanie M; Johnstone, Kim; Anderson, Derek M; Parrish, Christopher C; Rise, Matthew L

    2014-09-01

    For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod. PMID:24970595

  11. Recent progress in the discovery and development of stearoyl CoA desaturase inhibitors.

    PubMed

    Uto, Yoshikazu

    2016-05-01

    Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids from their saturated fatty acid precursors. SCD1 introduces a cis-double bond at the Δ9 position (between carbons 9 and 10) of stearoyl (C18:0) and palmitoyl-CoA (C16:0). SCD1 has been shown to be a crucial factor in lipid metabolism and body weight control. In addition, SCD1 inhibitors are claimed to be new treatments for various diseases, such as skin disorders, nonalcoholic steatohepatitis (NASH), hepatitis C virus (HCV), Alzheimer's disease, or cancer. This review aims to summarize the examples of the recently reported novel SCD1 inhibitors and to highlight the emerging areas of target indications that may hold promise for the development of SCD1 inhibitors. PMID:26344107

  12. Cloning and molecular characterisation of a Delta8-sphingolipid-desaturase from Nicotiana tabacum closely related to Delta6-acyl-desaturases.

    PubMed

    García-Maroto, Federico; Garrido-Cárdenas, José A; Michaelson, Louise V; Napier, Johnathan A; Alonso, Diego López

    2007-06-01

    Investigation on the absence of Delta(6)-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Delta(6)-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Delta(8)-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Delta(6)-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Delta(6)-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Delta(8)-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Delta(8)-desaturases. PMID:17325828

  13. Significant associations of stearoyl-CoA desaturase (SCD1) gene with fat deposition and composition in skeletal muscle.

    PubMed

    Jiang, Zhihua; Michal, Jennifer J; Tobey, David J; Daniels, Tyler F; Rule, Daniel C; Macneil, Michael D

    2008-01-01

    Gene expression studies in humans and animals have shown that elevated stearoyl-CoA desaturase (SCD1) activity is associated with increased fat accumulation and monounsaturation of saturated fatty acids in skeletal muscle. However, results of the two reported association studies in humans are inconsistent. In the present study, we annotated the bovine SCD1 gene and identified 3 single nucleotide polymorphisms (SNPs) in its 3'untranslated region (UTR). Genotyping these SNPs on a Wagyu x Limousin reference population revealed that the SCD1 gene was significantly associated with six fat deposition and fatty acid composition traits in skeletal muscle, but not with subcutaneous fat depth and percent kidney-pelvic-heart fat. In particular, we confirmed that the high stearoyl-CoA desaturase activities/alleles were positively correlated with beef marbling score, amount of monounsaturated fatty acids and conjugated linoleic acid content, but negatively with amount of saturated fatty acids. The inconsistent associations between human studies might be caused by using different sets of markers because we observed that most associated markers are located near the end of 3'UTR. We found that the proximity of the polyadenylation signal site is highly conserved among human, cattle and pig, indicating that the region might contain functional elements involved in posttranscriptional control of SCD1 activity. In conclusion, our cross species study provided solid evidence to support SCD1 gene as a critical player in skeletal muscle fat metabolism. PMID:18825276

  14. Evolution of Moth Sex Pheromone Desaturases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Moth sex pheromone communication has evolved to make use of complex blends of relatively simple long-chain fatty acid precursors. Species specificity is derived from the unique stereochemistry of double bonds introduced into exact locations along the hydrocarbon backbone of fatty acids, which are r...

  15. Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis.

    PubMed

    Michaelson, Louise V; Zäuner, Simone; Markham, Jonathan E; Haslam, Richard P; Desikan, Radhika; Mugford, Sarah; Albrecht, Sandra; Warnecke, Dirk; Sperling, Petra; Heinz, E; Napier, Johnathan A

    2009-01-01

    The role of Delta4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides. PMID:18978071

  16. Functional Characterization of a Higher Plant Sphingolipid Δ4-Desaturase: Defining the Role of Sphingosine and Sphingosine-1-Phosphate in Arabidopsis1[W][OA

    PubMed Central

    Michaelson, Louise V.; Zäuner, Simone; Markham, Jonathan E.; Haslam, Richard P.; Desikan, Radhika; Mugford, Sarah; Albrecht, Sandra; Warnecke, Dirk; Sperling, Petra; Heinz, E.; Napier, Johnathan A.

    2009-01-01

    The role of Δ4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Δ4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Δ4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Δ4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Δ4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Δ4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Δ4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides. PMID:18978071

  17. Desaturases -- Emerging Models for Understanding Functional Diversification of Diiron-Containing Enzymes

    SciTech Connect

    Shanklin, J.; Guy, J. E.; Mishra, G.; Lindqvist, Y.

    2009-07-10

    Desaturases and related enzymes perform O{sub 2}-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.

  18. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    PubMed

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants. PMID:27294968

  19. Genetic and Chemical Evaluation of Trypanosoma brucei Oleate Desaturase as a Candidate Drug Target

    PubMed Central

    Gualdrón-López, Melisa; Igoillo-Esteve, Mariana; Nguewa, Paul A.; Deumer, Gladys; Wallemacq, Pierre; Altabe, Silvia G.; Michels, Paul A. M.; Uttaro, Antonio D.

    2010-01-01

    Background Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. Methodology Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. Principal Findings Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC50 values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC50 values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential. Conclusions/Significance As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis. PMID

  20. Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells*

    PubMed Central

    Yee, Jennifer K.; Mao, Catherine S.; Hummel, Heidi S.; Lim, Shu; Sugano, Sharon; Rehan, Virender K.; Xiao, Gary; Lee, Wai-Nang Paul

    2008-01-01

    Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of stearate (18:0) to oleate (18:1n-9) and of palmitate (16:0) to palmitoleate (16:1), which are key steps in triglyceride synthesis in the fatty acid metabolic network. This study investigated the role of SCD1 in fatty acid metabolism in HepG2 cells using SCD1 inhibitors and stable isotope tracers. HepG2 cells were cultured with [U-13C]stearate, [U-13C]palmitate, or [1,2-13C]acetate and (1) DMSO, (2) compound CGX0168 or CGX0290, or (3) trans-10,cis-12 conjugated linoleic acid (CLA). 13C incorporation into fatty acids was determined by GC-MS and desaturation indices calculated from the respective ion chromatograms. FAS, SCD1, peroxisome proliferator-activated receptor α, and peroxisome proliferator-activated receptor γ mRNA levels were assessed by semiquantitative RT-PCR. The addition of CGX0168 and CGX0290 decreased the stearate and palmitate desaturation indices in HepG2 cells. CLA led to a decrease in the desaturation of stearate only, but not palmitate. Comparison of desaturation indices based on isotope enrichment ratios differed, depending on the origin of saturated fatty acid. SCD1 gene expression was not affected in any group. In conclusion, the differential effects of SCD1 inhibitors and CLA on SCD1 activity combined with the dependence of desaturation indices on the source of saturated fatty acid strongly support the compartmentalization of desaturation systems. The effects of SCD1 inhibition on fatty acid composition in HepG2 cells occurred through changes in the dynamics of the fatty acid metabolic network and not through transcriptional regulatory mechanisms. PMID:18599738

  1. Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

    DOEpatents

    Ohlrogge, J.B.; Cahoon, E.B.; Shanklin, J.; Somerville, C.R.

    1995-07-04

    The present invention relates to a process for producing lipids containing the fatty acid, petroselinic acid, in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a {omega}12 desaturase from another species which does normally accumulate petroselinic acid. 19 figs.

  2. Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

    DOEpatents

    Ohlrogge, John B.; Cahoon, Edgar B.; Shanklin, John; Somerville, Christopher R.

    1995-01-01

    The present invention relates to a process for producing lipids containing the fatty acid petroselinic acid in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a .omega.12 desaturase from another species which does normally accumulate petroselinic acid.

  3. The Crystal Structure of the Ivy delta4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

    SciTech Connect

    Guy,J.; Whittle, E.; Kumaran, D.; Lindqvist, Y.; Shanklin, J.

    2007-01-01

    The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the {Delta}{sup 4} desaturation of 16:0-ACP and the{Delta}{sup 9} desaturation of 18:0-ACP and further desaturates{Delta}{sup 9}-16:1 or {Delta}{sup 9}-18:1 to the corresponding {Delta}{sup 4,9} dienes. The crystal structure of the enzyme has been solved to 1.95{angstrom} resolution, and both the iron-iron distance of 3.2{angstrom} and the presence of a {mu}-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His{sup 227} coordination to Fe2 is lost, and the side chain of Glu{sup 224}, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.

  4. Plant fatty acid hydroxylases

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  5. Acyl-lipid desaturase 1 primes cold acclimation response in Arabidopsis.

    PubMed

    Chen, Mingjie; Thelen, Jay J

    2016-09-01

    Membrane fluidity change has long been suggested as the primary mechanism by which, plants adapt to cold stress, but the underlying molecular mechanisms are not completely established. In this study, we found that a knockout of acyl-lipid/CoA desaturase 1 gene (ADS1; EC 1.14.99) enhances freezing tolerance after cold acclimation (CA). Fatty acid composition analysis demonstrated that 18:1 content in ads1 mutant plants was 20% lower than in wild-type (WT) grown at 23°C. Lipidomics revealed that 34C-species of monogalactosyl diacylglycerol (MGDG) content in ads1 mutants were 3.3-14.9% lower than in WT. Lipid positional analysis identified 10% lower 18:1 fatty acid content at the sn-2 position of MGDG. The cytosolic calcium content in ads1 mutant plants was also approximately two-times higher than that of WT in response to cold shock. Each of these biochemical differences between WT and ads1 mutant disappeared after CA. Subcellular localization of C- and N-terminal enhanced-fluorescence-fusion proteins indicated that ADS1 localized exclusively to chloroplasts. These observations suggest that ADS1-mediated alteration of chloroplast membrane fluidity is required to prime a CA response, and is the upstream event of cytosolic calcium signaling. PMID:27062193

  6. Metabolomics Reveals that Hepatic Stearoyl-CoA Desaturase 1 Downregulation Exacerbates Inflammation and Acute Colitis

    PubMed Central

    Chen, Chi; Shah, Yatrik M.; Morimura, Keiichirou; Krausz, Kristopher W.; Miyazaki, Makoto; Richardson, Terrilyn A.; Morgan, Edward T.; Ntambi, James M.; Idle, Jeffrey R.; Gonzalez, Frank J.

    2008-01-01

    SUMMARY To investigate the pathogenic mechanism of ulcerative colitis, a dextran sulfate sodium (DSS)-induced acute colitis model was examined by serum metabolomic analysis. Higher levels of stearoyl lysophosphatidylcholine and lower levels of oleoyl lysophosphatidylcholine in DSS-treated mice compared to controls led to the identification of DSS-elicited inhibition of stearoyl-CoA desaturase 1 (SCD1) expression in liver. This decrease occurred prior to the symptoms of acute colitis and was well correlated with elevated expression of proinflammatory cytokines. Furthermore, Citrobacter rodentium-induced colitis and lipopolysaccharide treatment also suppressed SCD1 expression in liver. Scd1 null mice were more susceptible to DSS treatment than wild-type mice, while oleic acid feeding and in vivo SCD1 rescue with SCD1 adenovirus alleviated the DSS-induced phenotype. This study reveals that inhibition of SCD1-mediated oleic acid biogenesis exacerbates proinflammatory responses to exogenous challenges, suggesting that SCD1 and its related lipid species may serve as potential targets for intervention or treatment of inflammatory diseases. PMID:18249173

  7. Cloning and tissue distribution of a fatty acyl Δ6-desaturase-like gene and effects of dietary lipid levels on its expression in the hepatopancreas of Chinese mitten crab (Eriocheir sinensis).

    PubMed

    Yang, Zhigang; Guo, Zihao; Ji, Lianyuan; Zeng, Qitao; Wang, Yao; Yang, Xiaozhen; Cheng, Yongxu

    2013-06-01

    Fatty acyl Δ6-desaturase is the rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFAs) in vertebrates. In this report, a fatty acyl Δ6-desaturase-like cDNA was cloned from the hepatopancreas of Eriocheir sinensis (Chinese mitten crab) and characterized by performing rapid-amplification of cDNA ends. The 2278-bp long full-length cDNA encodes a polypeptide with 442 amino acids. Gene expression analysis via real-time quantitative polymerase chain reaction revealed that the fatty acyl Δ6-desaturase-like transcripts are widely distributed in various tissues, with high expression levels in the hepatopancreas and cranial ganglia. This study focuses on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Chinese mitten crab. A feeding trial was performed whereby crablets were fed for 238 d with four different diets: control diet without oil lipids (added with 3% basic lipid of the fundamental diets); fish oil diet (FO; added with 3% of the fundamental diets); soybean oil diet (SO; added with 3% of the fundamental diets); and FO/SO diet (1:1; added with 3% of the fundamental diets). The hepatopancreas of crabs sampled at 168 d and 238 d to determine the effects on fatty acyl Δ6-desaturase-like mRNA expression. The results show that the expression of fatty acyl Δ6-desaturase-like is higher in the hepatopancreas of crabs fed with SO diet than those fed with FO diet. Furthermore, gene expression increased by 2.45-fold in the hepatopancreas of crabs fed with SO after 238 d than those fed after 168 d but remained steady for those fed with FO after 238 d. PMID:23507625

  8. Rapid induction of microsomal delta 12(omega 6)-desaturase activity in chilled Acanthamoeba castellanii.

    PubMed

    Jones, A L; Lloyd, D; Harwood, J L

    1993-11-15

    The activity of microsomal delta 12-desaturase in Acanthamoeba castellanii was increased after growing cultures were chilled from the optimal growth temperature (30 degrees C) to 15 degrees C. This increase was detectable in microsomes isolated from organisms subjected to only 10 min chilling. The mechanism of induction was investigated. The increase in activity on chilling was greatly reduced when protein synthesis was blocked before the temperature shift. Thus the major mechanism for the induction of delta 12-desaturase is increased protein synthesis. delta 12-Desaturase activity was higher when assayed at 20 degrees C than when assayed at 30 degrees C, but these changes were not due to the increased solubility of O2 at 20 degrees C. The major substrate of delta 12-desaturase was found to be 1-acyl-2-oleoyl phosphatidylcholine. PMID:8250841

  9. Stereochemistry of 10-sulfoxidation catalyzed by a soluble delta9 desaturase

    SciTech Connect

    Tremblay, A.E.; Shanklin, J.; Tan, N.; Whittle, E.; Hodgson, D. J.; Dawson, B.; Buist, P. H.

    2010-03-21

    The stereochemistry of castor stearoyl-ACP 9 desaturase-mediated 10-sulfoxidation has been determined. This was accomplished by 19F NMR analysis of a fluorine-tagged product, 18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA. Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl substrate. These data validate the use of thia probes to determine the stereochemistry and cryptoregiochemistry of desaturase-mediated oxidations.

  10. Evolution of the Insect Desaturase Gene Family with an Emphasis on Social Hymenoptera

    PubMed Central

    Helmkampf, Martin; Cash, Elizabeth; Gadau, Jürgen

    2015-01-01

    Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes. PMID:25425561