• 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.

• 농약과학회지
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• 제14권1호
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• pp.72-77
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• 2010

3차원적 정량적인 구조-활성관계(3D-QSARs: CoMFA 및 CoMSIA)에 기초하여 phytoene desaturase (PDS)에 대한 O-(2-phenoxy)ethyl-N-aralkylcarbamate 유도체(1-15)의 제초성 평가를 위한 R-phenoxy 치환기들의 구조적인 요건들을 정량적으로 검토하였다. CoMFA 1 모델의 예측성 및 상관성($r^2_{cv.}=0.753$ 및 $r^2_{ncv.}=0.964$)이 나머지 모델들보다 높았다. 최적화된 CoMFA 1 모델로부터 PDS 저해활성은 O-(2-phenoxy)ethyl-N-aralkylcarbamate 유도체들의 입체장(44.0%), 정전기장(36.3%) 및 소수성장(19.6%)에 의존적이었다. CoMFA 등고도 분석결과, phenoxy 고리상 meta-와 para-위치에는 입체적으로 큰 치환기, meta-위치는 음하전, para-위치의 바깥 부분에는 양하전, ortho- 및 para- phenoxy 고리 중앙의 바깥 부분에는 친수성 치환기가 그리고 meta-위치에 소수성 R-치환기가 각각 도입될 경우에 PDS에 대한 저해활성이 증가할 것으로 예측되었다.

• 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.

• 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.

• Biomolecules & Therapeutics
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• 제20권5호
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• pp.470-476
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• 2012

Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol ($100{\mu}M$) for 24 hr induced cell death and cell cycle arrest in gastric cancer cells. Exposure to the combination of resveratrol and dimethylsphingosine (DMS) increased cytotoxicity, demonstrating that sphingolipid metabolites intensify resveratrol activity. Specifically, DHCer accumulated in a resveratrol concentration-dependent manner in SNU-1 and HT-29 cells, but not in SNU-668 cells. LC-MS/MS analysis showed that specific DHCer species containing C24:0, C16:0, C24:1, and C22:0 fatty acids chain were increased by up to 30-fold by resveratrol, indicating that resveratrol may partially inhibit DHCer desaturase. Indeed, resveratrol mildly inhibited DHCer desaturase activity compared to the specific inhibitor GT-11 or to retinamide (4-HPR); however, in SNU-1 cells resveratrol alone exhibited a typical cell cycle arrest pattern, which GT-11 did not alter, indicating that inhibition of DHCer desaturase is not essential to the cytotoxicity induced by the combination of resveratrol and sphingolipid metabolites. Resveratrol-induced p53 expression strongly correlated with the enhancement of cytotoxicity observed upon combination of resveratrol with DMS or 4-HPR. Taken together, these results show that DHCer accumulation is a novel lipid biomarker of resveratrol-induced cytotoxicity in human gastric cancer cells.

• Asian-Australasian Journal of Animal Sciences
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• 제24권6호
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• pp.757-765
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• 2011

Fatty acid composition of beef intramuscular tissue is an important trait because high proportions of mono-unsaturated fatty acid are related to favorable beef flavor. In this study, we investigated the effects of genetic factors, such as stearoyl-CoA desaturase (SCD), on beef carcass traits, including fatty acid composition, in the Hanwoo. Analysis of fatty acids in Hanwoo was performed using a breed raised in Gyeonbuk province (n = 395). Compared to the homozygote, the GA, CT, and CT genotypes of exon 5 in the SCD polymorphism showed a higher content of oleic acid (p<0.05) and higher contents of mono-unsaturated fatty acid (p<0.05) and marbling scores (p<0.05) in intramuscular fat. Results of haplotype analysis showed a significant presence of unsaturated fatty acids and marbling score in the $ht1^*ht2$ and $ht2^*ht2$ groups (p<0.05). Furthermore, haplotype effects more powerful than a single gene were also observed. These ht1 and ht2 types also showed a significant difference in unsaturated fatty acids and marbling score, affecting beef flavor in the Hanwoo groups. Therefore, it can be inferred that the ht1 and ht2 types might be valuable new markers for use in improvement of Hanwoo.

• 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.

• Plant Resources
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• 제1권1호
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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.

• Asian-Australasian Journal of Animal Sciences
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• 제18권4호
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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.

• 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.