Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.
Animals ; Caenorhabditis elegans/genetics* ; Fatty Acid Desaturases/genetics* ; Fatty Acids ; Fatty Acids, Omega-3 ; Mice ; Mice, Transgenic

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.
Fatty Acid Desaturases ; genetics ; metabolism ; Gene Expression Profiling ; Oleic Acid ; biosynthesis ; Phylogeny ; Plant Proteins ; genetics ; Seeds ; chemistry ; Soybeans ; classification ; enzymology ; genetics

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.
Acetyltransferases ; genetics ; metabolism ; Arachidonic Acid ; biosynthesis ; Docosahexaenoic Acids ; biosynthesis ; Eicosapentaenoic Acid ; biosynthesis ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acid Synthases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Transfection

Palmitoleic acid (16:1delta9), an unusual monounsaturated fatty acid, is highly valued for human nutrition, medication and industry. Plant oils containing large amounts of palmitoleic acid are the ideal resource for biodiesel production. To increase accumulation of palmitoleic acid in plant tissues, we used a yeast (Saccharomyees cerevisiae) acyl-CoA-delta9 desaturase (Scdelta9D) for cytosol- and plastid-targeting expression in tobacco (Nicotiana tabacum L.). By doing this, we also studied the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1delta9) and cis-vaccenic (18:1delta11) were significantly enhanced in the Scdelta9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1delta9 and 18:1delta11 in the Scdelta9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1delta9 levels. Our data indicate that yeast cytosolic acyl-CoA-delta9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1delta9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. The present study developed a new strategy for high accumulation of omega-7 fatty acids (16:1delta9 andl8:1delta11) in plant tissues by protein engineering of acyl-CoA-delta9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.
Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Monounsaturated ; metabolism ; Plants, Genetically Modified ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Tobacco ; genetics ; metabolism

BACKGROUNDA recent genome-wide association study in Caucasians revealed that three loci (rs174547 in fatty acid desaturase 1 (FADS1), rs2338104 near mevalonate kinase/methylmalonic aciduria, cobalamin deficiency, cblB type (MVK/MMAB) and rs10468017 near hepatic lipase (LIPC)) influence the plasma concentrations of high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG). However, there are few reports on the associations between these polymorphisms and plasma lipid concentrations in Chinese individuals. This study aimed to evaluate the associations between these three polymorphisms with HDL-C and TG concentrations, as well as coronary heart disease (CHD) susceptibility in Chinese individuals.

METHODSWe conducted a population-based case-control study in Chinese individuals to evaluate the associations between these three polymorphisms and HDL-C and TG concentrations, and also evaluated their associations with susceptibility to CHD. Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism assays and TaqMan genotyping assays.

RESULTSWe found significant differences in TG and HDL-C concentrations among the TT, TC and CC genotypes of FADS1 rs174547 (P=0.017 and 0.003, respectively, multiple linear regression). The CC variant of rs174547 was significantly associated with hyperlipidemia compared with the TT variant (adjusted odds ratio (OR)=1.71, 95% confidence intervals (CI): 1.16-2.54). The FADS1 rs174547 CC variant was also associated with significantly increased CHD risk compared with the TT and TC variant (adjusted OR=1.53, 95%CI: 1.01-2.31), and the effect was more evident among nonsmokers and females. The polymorphisms rs2338104 and rs10468017 did not significantly influence HDL-C or TG concentrations in this Chinese population.

CONCLUSIONrs174547 in FADS1 may contribute to the susceptibility of CHD by altering HDL-C and TG levels in Chinese individuals.

Aged ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Cholesterol, HDL ; blood ; Coronary Disease ; blood ; epidemiology ; genetics ; Fatty Acid Desaturases ; genetics ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Triglycerides ; blood

Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.
Amino Acid Sequence ; Cloning, Molecular ; Euglena gracilis ; enzymology ; Fatty Acid Desaturases ; biosynthesis ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism

Delta5-fatty acid desaturase is the key enzyme in synthesis of arachidonic acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic acid. The results indicated that high level expression of delta5-fatty acid desaturase, and the substrate conversion reached 45.9%.
Cloning, Molecular ; Diatoms ; enzymology ; genetics ; Fatty Acid Desaturases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.
Animals ; CHO Cells ; Caenorhabditis ; enzymology ; genetics ; Cricetinae ; Cricetulus ; Fatty Acid Desaturases ; genetics ; physiology ; Fatty Acids ; analysis ; Plasmids ; Polymerase Chain Reaction

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.
Chromatography, Gas ; Cloning, Molecular ; Fatty Acid Desaturases ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Soybeans ; enzymology ; genetics ; alpha-Linolenic Acid ; analysis ; biosynthesis ; genetics

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.
Cloning, Molecular ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Engineering ; methods ; Phylogeny ; gamma-Linolenic Acid ; biosynthesis