• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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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 Microbiology and Biotechnology
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• 제19권10호
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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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• 제30권4호
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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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• 제20권11호
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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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• 제26권6호
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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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• 제23권10호
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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.

• 한국식품영양과학회지
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• 제36권10호
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• pp.1279-1286
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• 2007

본 연구에서 4주령 ICR 수컷 마우스에 쇠기름과 어유에 단독 또는 0.5% CLA를 첨가한 식이로 4주동안 사육하여 혈장과 간의 지방산 조성과 DSD와 FADS2의 유전자 발현을 관찰한 결과는 다음과 같다. CLA는 C18:2와 경쟁적으로 지방산의 elongation과 desaturation에 참가하며, CLA의 첨가는 BTC군에서 C20:4수준을 높여주는 효과가 있었다. 또, 지방산의 ${\beta}$-산화에 의해 생성되기도 하는 E22:6은 BTC군에서 간의 C22:6의 함량이 BT군보다 증가하였다. DSD와 FADS2는 쇠기름섭취군(BT, BTC)에서 발현이 유의적으로 증가하였는데, 특히 D5D의 발현은 BTC군이 다른 군보다 유의적으로 증가하였다. 이에 대한 지방산 전환 또한 BTC 군이 C18:2로부터 C20:4로로 불포화하고 elongation하는 비율이 혈장과 간 모두에서 가장 높았다. 결론적으로 BT에 CLA를 첨가한 경우에 지방산 조성의 변화와 FADS2와 DSD의 발현을 증가시켜 지방산의 불포화도를 높여서 eicosanoids 합성과 항상성에 영향을 미치는 것으로 나타났으나 FO에 CLA첨가시에는 영향을 미치지 않는 것으로 나타났다.

• 한국육종학회지
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• 제50권4호
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• pp.463-471
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• 2018

들깨는 우리나라에서 재배되어온 대표적인 유지작물로 지질의 함량 중 알파리놀렌산의 함량이 60% 전후로 불포화도가 높아서 산패가 쉽게 일어나는 문제가 있다. 알파리놀렌산은 소포체 유래의 ${\Delta}15$ desaturase (FAD3)와 엽록체 유래의 ${\Delta}15$ desaturase (FAD7)에 의해서 합성된다. 엽록체 유래의 FAD7 유전자 발현의 손상없이 종자의 알파리놀렌산 함량을 낮추기 위해 소포체 유래 FAD3 유전자를 RNAi기법을 이용하여 발현을 억제하였다. 재배종인 엽실들깨의 배축을 이용하여 아그로박테리움 매개 형질전환법으로 제초제(바스타) 저항성을 가진 형질전환체 17개체를 획득하였다. 형질전환체는 0.3% (v/v) 바스타제초제를 이용하여 선발하였으며 Northern blot으로 FAD3 유전자의 발현이 억제되는 것을 확인하였다. 온실에서 수확한 12개체 종자 지방산 함량을 분석한 결과 알파리놀렌산 함량이 10-20% 2개체, 30-40% 7개체, 대조구와 비슷한 60%대 3개체를 획득하였다. 형질전환체의 $T_2$ 종자의 분리비와 지방산 조성을 분석한 결과 동형접합체 계통에서 6-10% 알파리놀렌산 함량을 보였으며 이형접합계통은 20-26% 알파리놀렌산 함량을 나타내어 동형으로 고정시 FAD3 유전자 발현이 상당히 강력히 억제됨을 확인할 수 있었다. 들기름의 지방산 중 알파리놀렌산 함량의 감소는 들깨의 산패를 방지하고 감마리놀렌산 등의 고가의 건강기능성 지방산 생산에 활용할 수 있을 것으로 기대된다.

• 한국응용곤충학회지
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• 제56권1호
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• pp.61-67
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• 2017

복숭아순나방(Grapholita molesta)은 두 가지 주요 성페로몬 성분(Z-8-dodecenyl acetate and E-8-dodecenyl acetate)을 갖고 있다. 이 성페로몬 성분의 생합성 과정 분석은 포화지방산의 10번 탄소에 이중결합을 합성하는 불포화효소($10{\Delta}$ DES)가 종 특이적 광학이성체 형성에 필수적이라고 제시하였다. 그러나 이 효소의 분자적 특징에 대해서 분석되지 않았다. 본 연구는 복숭아순나방 성페로몬 샘의 전사체에서 $10{\Delta}$ DES로 추정된 불포화효소(Gm-comp1575)의 단백질 기능 영역을 분석하였다. Gm-comp1575 유전자는 370개의 아미노산 서열 정보를 암호하고 있으며 분자량은 약 43.2 kDa 그리고 등전위점(pI)은 8.77로 추정되었다. 이 불포화효소는 4개의 막투과영역을 지니고 있으며, 6개의 탄수화물 결합 위치가 아미노 말단과 세포내 영역에서 갖는 것으로 추정되었다. 분자계통분석은 Gm-comp1575가 다른 종에서 알려진 $10{\Delta}$ DES와 유사성이 높은 것으로 밝혀졌다. Gm-comp1575 전사체는 암컷 성페로몬 샘 및 다른 복부 조직에서 발현되었다. 이 유전자 발현에 대한 RNA 간섭 처리는 처녀 암컷으로 하여금 사과원에서 수컷을 유인하는 능력을 크게 감소시켰다. 이러한 결과는 Gm-comp1575가 복숭아순나방의 성페로몬 생합성과 관련이 있는 유전자라고 제시하고 있다.

• KSBB Journal
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• 제20권2호
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• pp.93-99
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• 2005

불포화지방산의 생합성능이 뛰어나다고 알려진 Thraustochytrids 균주 중 26185를 대상으로 arachidonic acid (AA C20:4 n-6)를 포함하는 PUFA 생산에 관여하는 유전자의 하나로서 key enzyme인 desaturase를 보다 효율적이고 안정적인 방법으로 cloning한 후 결과물을 분석하였다. 이를 위해 실험균주인 26185 균주를 대상으로 효율적인 전체 nucleic acids 추출법을 개발하였으며 관련된 기법의 활용을 통해 일반적으로 활용이 가능한 효과적이 방법임을 확인하였다. 확보된 유전체를 통한 direct PCR의 결과물을 기존에 알려진 cDNA 합성방법에 의한 결과물과 비교하였을 때 동일한 결과물임을 확인하였다. 이는 Thraustochytrids 관련 균주로부터 지방산 생합성에 관련된 효소군을 탐색하는 방법이 cDNA 합성방법에 의하지 않고 보다 직접적으로 진행될 수 있음을 제안할 수 있는 하나의 결과물로 생각되어진다. 획득된 유전자를 같은 진핵세포인 P. pastoris를 숙주로 하여 유전자를 도입하고 활성을 재현성 있게 규명함으로써 인공진화 혹은 재조합균체의 개발이 가능하다는 사실을 부분적으로 제시할 수 있었다.