• 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 Microbiology and Biotechnology
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• v.20 no.11
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• pp.1555-1562
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• 2010

Development of the strain and the fermentation process of Hansenula polymorpha was implemented for the production of ${\gamma}$-linolenic acid ($GLA,\;C18:3{\Delta}^{6,9,12}$), an n-6 polyunsaturated fatty acid (PUFA) that has been reported to possess a number of health benefits. The mutated ${\Delta}^6$-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without the utilization of methanol, a high-cell-density culture of the yeast recombinant carrying the ${\Delta}^6$-desaturase gene was then achieved by fed-batch fermentation under glycerol-limited conditions. As a result, high levels of the ${\Delta}^6$-desaturated products, octadecadienoic acid ($C18:2{\Delta}^{6,9}$), GLA, and stearidonic acid ($C18:4{\Delta}^{6,9,12,15}$), were accumulated under the derepression conditions. The GLA production was also optimized by adjusting the specific growth rate. The results show that the specific growth rate affected both the lipid content and the fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates tested, the highest GLA concentration of 697 mg/l was obtained in the culture with a specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor ${\Delta}^6$-desaturase gene was similar to that of blackcurrant oil, with both containing similar proportions of n-3 and n-6 essential fatty acids.

• 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 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.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.4
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• pp.562-566
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• 2005

This study was conducted to investigate the developmental relationship between fatty acid composition in different lipid fractions and stearoyl-CoA desaturase (SCD) gene expression in steer muscles during growth. Twenty Hanwoo steers were used at 6, 12, 18, 24 and 30 months of age. Fatty acid composition and SCD mRNA level were analyzed. In the total lipid fraction, developmental profiles of C18:1, as the product of SCD enzyme, and SCD mRNA level were significantly increased between 6 months and 12 months of age. During this period, the percentage of C18:1 increased from 31.9% to 49.5% in the total lipid. The increased C18:1 level was maintained until 30 months of age within the range of 44.8- 49.9%. In contrast, the C18:0 composition decreased with age and this decrease was compensated by the increase of the C18:1. However, the sum of C18:0 and C18:1 was changed before and after 12-month old by a 20% increase. Unlike the C18 fatty acids, the C16 fatty acids such as C16:0 and C16:1 did not show a consistent change with age in steers' muscle. On the other hand, C18:2 proportion as a major polyunsaturated fatty acid in muscle was significantly reduced from 21.1% at 6 months of age to 4.4% at 12-months old and then this reduced level was maintained until 30 months within the range of 7.4-11.4%. As in the C18:1 composition during early stages, a 2-fold significant increase was observed in the $\Delta^9$-desaturase index of C18 fatty acid as a measure of SCD activity, but not in that of C16 fatty acid. Also, the steady-state level of SCD mRNA reached a peak at 12 months of age. Thus, the positive relationship between the C18:1 composition and the $\Delta^9$-desaturase (SCD enzyme) index of C18 fatty acid or SCD mRNA level was demonstrated during growth, but the negative relationship between the C18:2 composition and the above three indices was demonstrated at the same time, indicating that the sharp induction of SCD mRNA may be closely related to the dramatic reduction of C18:2, which is known as a suppressor of SCD gene expression during growth.

• The Korean Journal of Food And Nutrition
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• v.20 no.3
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• pp.245-252
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• 2007

This study investigated the effects of conjugated linoleic acid(CLA) on the fat deposition, triglyceride levels and the expression of stearoyl-CoA desaturase 1(SCD1) in the livers of male ICR mice that were fed with either soybean oil or beef tallow supplemented with CLA. Mice weighing $25{\sim}30$ g were divided into four groups; soybean oil(SBO), and SBO supplemented with 1% CLA(SBOC), beef tallow(BT) and BT supplemented with 1% CLA(BTC). Each group consisted of 10 mice that were fed the experimental diets for 4 weeks. The experimental diets consisted of 64% carbohydrate, 20% protein, and 16% fat in terms of their contributions to total calories. All other nutrients were identical in the diets. Triglyceride measurements were completed using a kit. Fatty acid compositions were analyzed in the liver using gas chromatography. The levels of SCD1 expression were analyzed by RT-PCR in the liver. No significant differences were found for food intake level, body weight and food efficiency among the experimental groups. However, the weights of epididymal fat pads and plasma triglyceride levels were significantly lower in SBOC and BTC(p<0.05) compared to the SBO and BT groups. These effects were similar in the CLA supplemented groups. The expression level of SCD1 gene and ${\Delta}9$ desaturase index were not significantly different, regardless of the fat used for CLA supplementation. Based on these results, addition of CLA showed decreasing effects on the fat depots weight and the concentration of triglyceride regardless of the fat sources. The SCD1 gene expression and ${\Delta}9$ desaturase index were not influenced by the types of fats with respect to the CLA effects.

• Journal of Crop Science and Biotechnology
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• v.10 no.4
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• pp.201-210
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• 2007

Soybean [Glycine max(L.) Merr.] oil is versatile and used in many products. Modifying the fatty acid profile would make soy oil more functional in food and other products. The ideal oil with the most end uses would have saturates(palmitic + stearic acids) reduced from 15 to < 7%, oleic acid increased from 23 to > 55%, and linolenic acid reduced from 8 to < 3%. Reduced palmitic acid(16:0) is conditioned by three or more recessive alleles at the Fap locus. QTLs for reduced palmitic acid have mapped to linkage groups(LGs) A1, A2, B2, H, J, and L. Genes at the Fad locus control oleic acid content(18:1). Six QTLs($R^2$=4-25%) for increased 18:1 in N00-3350(50 to 60% 18:1) explained four to 25% of the phenotypic variation. M23, a Japanese mutant line with 40 to 50% 18:1 is controlled by a single recessive gene, ol. A candidate gene for FAD2-1A can be used in marker-assisted breeding for high 18:1 from M23. Low linolenic acid(18:3) is desirable in soy oil to reduce hydrogenation and trans-fat accumulation. Three independent recessive genes affecting omega-3 fatty acid desaturase enzyme activity are responsible for the lower 18:3 content in soybeans. Linolenic acid can be reduced from 8 to about 4, 2, and 1% from copies of one, two, or three genes, respectively. Using a candidate gene approach perfect markers for three microsomal omega-3 desaturase genes have been characterized and can readily be used in for marker assisted selection in breeding for low 18:3.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• Korean Journal of Breeding Science
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• v.50 no.4
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• pp.463-471
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• 2018

Perilla is an oilseed crop cultivated in Korea since ancient times. Due to the high ${\alpha}-linolenic$ acid content in perilla, perilla seed oil can easily become rancid. ${\alpha}-Linolenic$ acid is synthesized by two enzymes, endoplasmic reticulum-localized ${\Delta}15$ desaturase (FAD3) and chloroplast-localized ${\Delta}15$ desaturase (FAD7) in vivo. In order to lower the ${\alpha}-linolenic$ acid content of the seed oil without disturbing plant growth, we tried to suppress the expression of only the FAD3 gene using RNA interference, whilst maintaining the expression of the FAD7 gene. Seventeen transgenic plants with herbicide ($Basta^{TM}$) resistance were obtained by Agrobacterium-mediated transformation using hypocotyls of perilla plants. The transgenic plants were firstly confirmed by treatment with 0.3% (v/v) $Basta^{TM}$ herbicide, and the expression of FAD3 was measured by Northern blot analysis. The ${\alpha}-linolenic$ acid content was 10-20%, 30-40%, and 60% in two, seven, and three of the twelve $T_1$ transgenic perilla plants which had enough seeds to be analyzed for fatty acid composition, respectively. Analysis of the fatty acid composition of $T_2$ progeny seeds from $T_1$ plants with the lowest ${\alpha}-linolenic$ acid content showed that the homozygous lines had 6-10% ${\alpha}-linolenic$ acid content and the heterozygous lines had 20-26% ${\alpha}-linolenic$ acid content. It is expected that the reduction in ${\alpha}-linolenic$ acid content in perilla seed oil will prevent rancidity and can be utilized for the production of high-value functional ingredients such as high ${\gamma}-linolenic$ acid.

• 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.