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

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

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

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

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

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. P34H, a member of the short-chain dehydrogenase/reductase(SDR) superfamily, is acquired on the acrosomal cap of human spermatozoon during its maturation arising within epididymis. P34H has been shown to be involved in sperm-zona pellucida interaction. Research revealed that the occurrence of low concentration of sperm protein P34H were significant amongst the idiopathic infertile male population and P34H protein could also be considered as a marker of epididymal sperm maturation in human. Therefore the level of sperm protein P34H is proposed to be a auxiliary diagnostic tool for male infertility. This paper reviews the molecular properties and regulation of the expression of P34H and its association with male reproduction.
Acyl-CoA Dehydrogenase ; Fatty Acid Desaturases ; chemistry ; Gene Expression ; Humans ; Male ; Proteins ; genetics ; physiology ; Sperm Maturation ; physiology ; Sugar Alcohol Dehydrogenases

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

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

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

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 maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase.
Agrobacterium tumefaciens ; genetics ; Fatty Acid Desaturases ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mortierella ; enzymology ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Polymerase Chain Reaction ; Tobacco ; genetics ; metabolism ; gamma-Linolenic Acid ; biosynthesis