• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.116-121
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• 2010

Long-chain polyunsaturated fatty acids (LC-PUFA) promote the development of brain and vision of the fetus, relieve inflammation, inhibit oral dysplasia of rumor cell, decrease the incidence of cardiovascular disease and regulate arrhythmia. ${\Delta}-6$ desaturase is the rate-limited enzyme in the desaturation process. This study reports the cloning, characterization and tissue expression of a ${\Delta}-6$ desaturase gene in the chicken. PCR primers were designed based on the predicted sequence of chicken ${\Delta}-6$ desaturase (accession number: XM421053) and used to isolate a cDNA fragment of 1,323 bp from chicken liver. Based on the 1,323 bp fragment an EST (BI390105) was obtained by BLAST. The EST and 5'nd of the 1,323 bp fragment were partially overlapped. Gene specific primers derived from the EST were used for amplification of the 5'nd. Another gene-specific primer derived from the 1,323 bp fragment was used for amplification of the 3'nd by 3'ACE. Then the three overlapping cDNA sequences obtained were assembled with DNAMAN software and a full-length ${\Delta}-6$ desaturase of 2,153 bp was obtained. The full-length cDNA contained an ORF of 1,335 bp with a 5'ntranslated region of 147 nucleotides followed by an ATG initiation codon. Stop codon TGA was at position 1,481-1,483 bp. The deduced amino acids shared an homology above 77% with bovine, mice, orangutan, rat and human. The protein sequence had three histidine-rich regions HDFGH (HisI region), HFQHH (HisII region) and HH (HisIII region), a cytochrome $b_{5}$-like domain containing a heme-binding motif and two transmembrane domains. Sequence analysis of the chicken genomic DNA revealed that the coding sequence of chicken ${\Delta}-6$ desaturase included 12 exons and 11 introns. Semi-quantitative RT-PCR showed that the ${\Delta}-6$ desaturase expression levels were in turn liver, spleen, pancreas, lung, breast muscle, heart, and abdominal fat. The expression of ${\Delta}-6$ desaturase in liver was significantly higher than that in breast muscle (p<0.01). The expression of ${\Delta}-6$ desaturase in lung was significantly higher than that in abdominal fat (p<0.01). This is the first clone of chicken ${\Delta}-6$ desaturase.

• Journal of Nutrition and Health
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• v.30 no.4
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• pp.442-450
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• 1997

Conjugated dienoic derivatives of linoleic acid(CLA) are a mixture of positional and geometric isomers of linoleic acid(LA). We previously found that CLA changes the fatty acid profile in chicken eggs and serum by decreasing monounsaturated fatty acids. Studies were conducted to explore the effects of CLA on fatty acid composition. Rabbits were fed a semisynthetic diet with or without CLA(0.5g CLA/rabbit/day) for 22 weeks. Compared to the control, rabbits fed CLA had significantly lower monounsaturated fatty acid levels(palmitoleic acid Cl6 : 1 by 50% and oleic acid Cl8 : 1, by 20%) in plasma lipids. We found similar differences in fatty acid composition in the liver and the aorta. The inhibitory effect of CLA on $\Delta$9 desaturation was confirmed in a human hepatoma cell line, Hep G2. CLA significantly decreased $\Delta$9 desaturation in 4-5 hours as shown by an increase in the ratio of Cl6 : 0 to C 16 1, This is apparently due to a decrease in $\Delta$9 desaturase(stearoyl-CoA desaturase, SCD) activity ; it was decreased more than 50%. These results, along with our previous findings, indicate that CLA is an inhibitor of $\Delta$9 desaturase in the liver.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1098-1102
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• 2009

Gamma-linolenic acid (GLA, C18:3 ${\Delta}^{6,9,12}$) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 ${\Delta}^{9,12}$) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.

• KSBB Journal
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• v.26 no.6
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• pp.557-560
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• 2011

It has been known that Δ-desaturase (TAD5) in the biosynthetic pathway of long chain polyunsaturated fatty acids of Thraustochytrium aureumis responsible for the conversion of di-homo-${\gamma}$-linolenic acid (C20：4) into arachidonic acid (C20：4). The genetic sequence analysis on TAD5 of Thraustochytrium aureum ATCC34304 used in this study showed that it has two amino acid changes when compared to that of Thraustochytrium aureum TAD5 first reported in 2003. Accordingly, Thraustochytrium aureum ATCC34304 TAD5 was named TAD5_1. TAD5_1-inserted methylotropic Pichia pastoris was prepared and then cultured with a precursor fatty acid, di-homo-${\gamma}$-linolenic acid. GC analysis confirmed that a certain amount of the precursor fatty acid was converted into arachidonic acid. In this study, not only a recombinant Pichia pastoris with the typical activity of ${\Delta}5$-desaturase which plays an essential role in the biosynthesis of LCPUFAs was successfully made but also the preparationpotential of a recombinant Pichia pastoris strain which may synthesize eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that are important in maintaining and improving human's brain function was proposed.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1413-1421
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• 2013

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, $C20:5{\omega}-3$) and docosahexaenoic acid (DHA, $C22:6{\omega}-3$) that are important to human health. Here, we report a functional characterization of a ${\Delta}4$-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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.10
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• pp.1279-1286
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• 2007

We investigated the effects of conjugated linoleic acid (CLA) on the fatty acid composition in the plasma and liver, and the expressions of delta-5 desaturase (D5D) and fatty acid desaturase2 (FADS2) genes in ICR male mice using two different sources of fats in the diets. The experimental groups were divided into four groups: beef tallow (BT) and fish oil (FO), BT with CLA supplementation (BTC), and FO with CLA supplementation (FOC) groups. Ten mice in each group were fed with the experimental diets for 4 weeks. All mice were fed experimental diets containing 12% of total dietary fat (w/w) either with or without 0.5% CLA (w/w). Fatty acid compositions were analyzed in the plasma and liver using gas chromatography. The levels of D5D and FADS2 expression were analyzed by RT-PCR in the liver The results showed that CLA participates competitively with C18:2 in the elongation and desaturation processes, leading to significant increase in the levels of C20:4 and C22:6 in BTC group (p<0.05). The expression levels of D5D and FADS2 were higher in BT and BTC group than those of FO and FOC group. In particular, the expression of D5D gene was greatly upregulated in BTC group. Furthermore, the conversion ratios from C18:2 to C20:4 in the liver were higher in BTC group than those in other groups. Thus our results suggest that increased expressions of DSD and FADS2 genes may be responsible for the enhanced CLA effects on the desaturation in the BT containing saturated fatty acids rather than the FO rich in n-3 PUFA.

• KSBB Journal
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• v.20 no.2 s.91
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• pp.93-99
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• 2005

Polyunsaturated fatty acids, that is PUFAs, are important constituents of membranes particularly found in the retina and central nervous system. In microorganism-based PUFAs biosynthesis, the genus Thraustochytrids is well evaluated for their potential as a promising candidate in the practical production of PUFAs, such as AA and DHA. In this study, we attempted to optimize a method of total nucleic acid extraction from this microorganism as a preliminary experiment. Using the extracted nucleic acid and degenerated primers for direct PCR, we isolated a $\Delta5$ desaturase gene that contained 1320-nucleotide and encoded 439 amino acids. This gene exhibited an expected function, when expressed in P. pastoris in the presence of appropriate exogenous substrate, as an evidence for $\Delta5$ desaturase activity (conversion of DGLA to AA). These results and information could provide a basis for the construction of engineered strains suitable for the practical production of PUFAs.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.399-399
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• 2005

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.507-514
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• 2010

Peanut(Arachis hypogaea L.) is one of the major oilseed crops. The peanut oil consists of palmitic, oleic and linoleic acids, which are present at levels of 10%, 36-67% and 15-43%, respectively. High oleate mutant of peanut F435 contains 80% oleate and as little as 2% linoleate in seed oil. Previous study indicated that delta 12 fatty acid desaturase is a major enzyme controlling the oleate content in seeds of oilseed crops. F435 sequence alignment of their coding regions disclosed that an extra A(adenine) was inserted at the position +2,823 bp of delta 12 fatty acid desaturase gene. This study was to develop molecular marker (SNP marker) co-segregating with the high oleate trait. Chopyeong ${\times}$ F435 $F_2$ 41 population were investigated using molecular marker and fatty acid assay (NIR and gas chromatography). Finally, this marker segregates Chopyeong type 26 lines, heterotype 9 lines and F435 type 6 lines. These results in our study suggested that SNP marker conform fatty acid assay.

• Plant Resources
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• v.1 no.1
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• pp.33-37
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• 1998

Carotenoid compounds in embryos of wild-type(WT) and viviparous mutants of maize(Zea mays L.) were analyzed using high performance liquid ehromatography (HPLC) with a photodiode array detector. Zeaxanthin accumulates in WT embryos as the major carotenoid. Phytoene accumulates in vp2 and vp5. Phytofluene in w3 and ${\xi}$-carotene in the vp9 mutant embryos. This indicates that the vp2 and vp5 mutants impair phytoene desaturase from 15-cis-phytoene to 15-cis-phytofluene. The w3 mutant has neither an isomerase from 15-cis-phytofluene to all-trans-phytofuene nor phytofluene desaturase from phytofluene to ${\xi}$-carotene. The vp9 mutant does not have the ${\xi}$-carotene desaturase from ${\xi}$-carotene to lycopene. Our analysis shows that the terminal carotenoid. ${\gamma}$-carotene(${\beta},{\Psi}$-carotene), accumulates in the vp7 mutant embryos. The ${\varepsilon}$-carotene(${\varepsilon},{\varepsilon}$-carotene), a product of ${\delta}$-carotene(${\varepsilon},{\Psi}$-carotene) in some plants, however, has not been found in maize embryos. The vp7 mutant impairs a cyclization step from ${\gamma}$-carotene to both ${\beta}$-carotene and ${\alpha}$-carotene. We suggest that monocyclic ${\gamma}$-carotene is the sole precursor of both bicyclic ${\beta}$-carotene(${\beta},{\beta}$-carotene) and ${\alpha}$-carotene(${\beta},{\varepsilon}$-carotene) in maize.