• 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.

• 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.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 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.

• 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.

• 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.

• Journal of the Korean Applied Science and Technology
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• v.13 no.3
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• pp.15-24
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• 1996

Essentiality was proposed in the field of lipid by Burr and Burr in 1929. When rats were raised on the fat-free diet, their growth retarded and their skin and tails showed the characteristic deficient symptoms, which were relieved by the addition of ${\omega}6(n-6)$ polyunsaturated fatty acids as linoleic(LA) and arachidonic(AA) acids to the basal diet. LA is dehydrogenated to ${\gamma}-linolenic$ acid(GLNA) by ${\Delta}6$ desaturase, then GLNA is 2 carbon chain elongated by elongase to $dihomo-{\gamma}-linolenic$ acid(DGLNA), which is desaturated by ${\Delta}5$ desaturase to AA. These acids are called LA family or ${\omega}6(n-6)$ polyunsaturated fatty acids(PUFA). ${\alpha}-Linolenic$ acid(ALNA) is converted through the series of desaturation and elongation steps to docosahexaenic acid(DHA) via eicosapentaenoic acid(EPA). These acids belong to ALNA family or ${\omega}3(n-3)$PUFA. Human who consume large amounts of EPA and DHA, which are present in fatty fish and fish oils, have increased levels of these two fatty acids in their plasma and tissue lipids at the expense of LA and AA. Alternately, vegetarians, whose intake of LA in high, have more elevated levels of LA and AA and lower levels of EPA and DHA in plasma lipids and in cell membranes than omnivores. AA and EPA are metabolized to substances called eicosanoids. Those derived form AA are known as prostanocids(prostaglandins and prostacyclins) of the 2-types and leukotrienes of the 4-series, whereas those derived from EPA are known as prostanoids of the 3-types and leukotrienes of the 5-series. DGLNA is a precursor of the 1-types of prostaglandins. The metabolites of AA and EPA have competitive functions. Ingestion of EPA from fish or fish oil replaces AA from membrane phospholipids in practically all cells. So this leads to a more physiological state characterized by the production of proatanoids and leukotrienes that have antithrombic, antichemotactic, antivasoconstrictive and antiinflammatory properties. It is evident that ${\omega}3$ fatty acids can affect a number of chronic diseases through eicosanoids alone.

• Proceedings of the EASDL Conference
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• 2004.10a
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• pp.13-30
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• 2004

There are two groups of significant functional constituents in sesame seeds on the whole; one is the vegetable oils and another is the anti-oxidative compounds. However, although high amounts of major fatty acids are synthesized in sesame seeds, their composition is unfavorable because the contents of alpha- and gamma-linolenic acid, the essential fatty acids, are very low or do not produced in sesame seeds. So, to increase these fatty acids in sesame seeds, one strategy is to overexpress their genes, ${\omega}$-3 fatty acid desaturase for alpha-linolenic acid and delta-6 fatty acid desaturase for gamma-linolenid acid, in them. Another molecular target is to enhance alpha-tocopherol, vitamin E, because its content is very low in sesame seeds. The enzyme, gamma-tocopherol methyltransferase, catalyzes the conversion of gamma-tocophero to alpha-tocopherol. Overexpression of this enzyme in sesame seeds could be also a good molecular breeding target. Reduction of phytic acid is also another molecular target in sesame seeds because phosphorus pollution may be caused by its high content in sesame seeds. Accordingly, to do so, one of target enzymes could be myo-inositol 1-phosphate synthase which is a key regulatory enzyme in the pathway of phytic aicd biosyntheses. In this lecture, a molecular strategy for development of value-added sesame crop is described in association with some results of our experiments involved in the molecular characterizations of the genes mentioned above.