长链多不饱和脂肪酸的替代来源——转基因油料作物

长链多不饱和脂肪酸(LCPUFA)对人体健康和发育具有极为重要的作用.由于海洋鱼类资源过度捕捞和环境污染的加剧,长链多不饱和脂肪酸的来源日益枯竭.利用转基因油料作物生产LCPUFA,特别是DHA和EPA成为目前研究的热点.归纳了LCPUFA合成代谢途径相关基因的最新研究进展,描述了利用植物基因工程合成长链多不饱和脂肪酸(特别是EPA和DHA)取得的主要成果,讨论了影响转基因油料作物合成DHA和EPA的关键因素,最后对利用转基因油料作物合成DHA和EPA的研究前景进行了展望.     

饲料中不同EPA和DHA含量对黄鳝脂类代谢、生长及繁殖性能的影响

为研究EPA和DHA对黄鳝脂类代谢、生长及繁殖性能的影响, 用添加有EPA和DHA比例相同(EPA/DHA=0.35)但水平不同(EPA和DHA总量分别为0.00%、0.35%、0.70%)的3种等氮等脂肪饲料, 在常温(2230 ℃)下, 分别喂养平均体重为(21.710.54) g的黄鳝7周, 分析黄鳝脂类代谢和生长性能, 然后每组取15尾继续养殖至性腺发育成熟用于繁殖性能分析。结果表明:在饲料中添加0.70%的EPA和DHA 能促进黄鳝生长, 显著提高黄鳝的特定生长率(SGR)及肌肉粗蛋白含量, 降低肝体指数(HSI)及肌肉、肝脏中的粗脂肪含量(P0.05), 显著提高亲鳝的性体指数(GSI)、产卵量、孵化率、幼苗的存活指数(SAI)(P0.05)。添加0.35%的EPA和DHA能显著降低肝脏中粗脂肪含量(P0.05)。综合分析表明:EPA和DHA能促进黄鳝的脂类代谢, 显著提高其生长及繁殖性能; 饲料中添加0.70%的EPA和DHA较为适宜。       

真菌发酵生产鱼油特征脂肪酸

DHA和EPA是鱼油的特征脂肪酸,具有调整血脂、改善大脑功能和维持正常视力等重要作用.一直以来,其来源主要是海产鱼油.近年研究发现许多微生物的脂质中也含有DHA和EPA,特别是一些低等真菌含量甚为丰富,利用其发酵生产DHA和EPA替代鱼油资源具有广阔的前景.阐述了能产生EPA,DHA的真菌及其合成途径和发酵影响因素.     

真菌发酵生产EPA及DHA影响因素的研究进展

对真菌发酵生产EPA及DHA的影响因素进行综述 ,介绍了菌种、碳源、氮源、C/N比、pH值、温度、发酵时间、通气量、代谢途径的调控、种龄和接种量等因素对EPA及DHA产量的影响。     

四个脂肪酸合成酶基因在哺乳动物细胞中的超表达提高长链多不饱和脂肪酸生物合成效率

DHA(22:6n-3)、EPA(20:5n-3)和ARA(20:4n-6)三种长链多不饱和脂肪酸在生物体内活性最强,它们在促进大脑发育和功能维持以及在预防和治疗心血管疾病、炎症、癌症等多种疾病方面有着重要作用。然而,尽管哺乳动物体内有完整的长链多不饱和脂肪酸合成酶系,但哺乳动物合成这些长链多不饱和脂肪酸的效率很低而主要依赖于食物获取。本研究应用转基因方法,将哺乳动物来源的Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶这4种酶的编码基因构建成为一个多基因表达载体,然后转染哺乳动物细胞HEK293T,实现了4个目的基因的超表达,再通过气质联用(GC-MS)分析证实了DHA、EPA和ARA等长链多不饱和脂肪酸的合成效率及水平显著增加,DHA的水平更是提高了2.5倍。由此可见,哺乳动物具有某种抑制长链多不饱和脂肪酸高水平合成的机制,但通过Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶的超表达,能够打破哺乳动物这种抑制机制,从而显著提高DHA、EPA、ARA等的合成水平。同时,本研究的思路也为在转基因动物中生产长链多不饱和脂肪酸提供了重要的启示。     

ISAAA信息

<正>转基因亚麻产生大量OMEGA-3脂肪酸洛桑研究所的研究人员成功地对亚麻(Camelina sativa)种子的新陈代谢途径进行了改造,并使得其十二碳五烯酸(EPA)和二十二碳六烯酸(DHA)的产量达到了12%和14%,这个组成比例     

长链多不饱和脂肪酸EPA、DHA的基因工程研究进展

长链多不饱和脂肪酸(long chain polyunsaturated fatty acid,LCPUFA)对维持人体健康具有重要作用,对其需求逐年增加,但是由于环境污染与渔业资源的下滑,有限的鱼油资源越来越不能满足人们需求。运用现代生物技术人们已相继分离了多个LCPUFA合成相关基因,并阐明了多条LCPUFA合成代谢途径。通过转基因技术在高等植物中成功合成了对人体健康十分重要的长链多不饱和脂肪酸,尤其是二十碳五烯酸(EPA),二十二碳六烯酸(DHA)。综述了LCPUFA的合成途径及转基因研究的最新进展,分析合成LCPUFA存在的问题及解决方法,并对未来多不饱和脂肪酸EPA,DHA的基因工程研究进行展望。     

ISAAA信息

<正>转基因亚麻产生大量OMEGA-3脂肪酸洛桑研究所的研究人员成功地对亚麻(Camelina sativa)种子的新陈代谢途径进行了改造,并使得其十二碳五烯酸(EPA)和二十二碳六烯酸(DHA)的产量达到了12%和14%,这个组成比例     

ω-3多不饱和脂肪酸抗肿瘤新途径——抑制具核梭杆菌黏附宿主细胞

【背景】大量文献报道ω-3多不饱和脂肪酸尤其是二十二碳六烯酸(Docosahexaenoic Acid,DHA)与二十碳五烯酸(Eicosapentaenoic Acid,EPA)具有抗肿瘤作用,但是其抗肿瘤机制还不够完善。【目的】探究ω-3多不饱和脂肪酸、具核梭杆菌以及结直肠癌三者之间的关联。【方法】在检测二十二碳六烯酸、二十碳五烯酸、α-亚麻酸(α-Linolenic Acid,ALA)等ω-3多不饱和脂肪酸对人结直肠腺癌细胞Caco-2、正常结肠上皮细胞NCM460生长影响的基础上,检测DHA等3种多不饱和脂肪酸对具核梭杆菌黏附人体细胞以及Fap2、FadA、RadD等具核梭杆菌毒力关键基因表达的影响。【结果】30μg/mL的DHA、EPA、ALA对Caco-2生长抑制分别为9.09%、4.95%、7.52%,而对NCM460生长抑制达31.15%、25.48%、29.11%,而且相关抑制作用仅具有浓度依赖性而无时间依赖性。经30μg/mL的DHA、EPA、ALA预处理的具核梭杆菌黏附Caco-2细胞的能力分别下降81.04%(P=0)、93.63%(P=0)和68.63%(P=0);而共培养时加入DHA、EPA、ALA对具核梭杆菌黏附Caco-2细胞的能力没有显著影响。同时,30μg/mLDHA处理导致F.nucleatum的Fap2基因显著下降10.22%(P=0.027);30μg/mL EPA处理导致FadA、Fap2基因分别显著下降23.49%(P=0)、15.09%(P=0.003);30μg/mL ALA处理导致FadA基因显著下降26.75%(P=0.012)。【结论】综合上述实验结果以及DHA、EPA、ALA仅能短时间抑制具核梭杆菌生长等文献报道,我们认为,DHA、EPA等ω-3多不饱和脂肪酸并非简单地直接杀伤或抑制肿瘤细胞和F.nucleatum;抑制FadA、Fap2等黏附相关基因表达,降低F.nucleatum黏附宿主细胞能力是其抗肿瘤作用的关键组成部分。ω-3多不饱和脂肪酸等活性物质对F.nucleatum等在结直肠肿瘤发生、发展中发挥重要作用的肠道细菌的影响与机制应深入开展研究。     

EPA DHA对体内脂质代谢影响的研究进展

EPA和DHA是人体所需的重要的不饱和脂肪酸,发挥多种重要的生理作用,影响人类的生长发育与身体健康。近年来,人们的健康保健意识逐渐加强,更为关注EPA和DHA的保健营养价值。因此,EPA和DHA生物制品的提取开发成为了食物资源开发利用的热点问题。作为有较疗效好、安全性高的营养品,培养富含EPA、DHA的生物制品会有一个光明的前景。本文介绍了EPA和DHA的生物学特性、来源,并着重阐述其在机体脂质代谢中的作用。     

Recombinant production of omega‐3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1

Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Dietary Effects of Eicosapentaenoic and Docosahexaenoic Acid Esters on Lipid Metabolism and Immune Parameters in Sprague-Dawley Rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB₄ productivity and an increase of LTB₅ productivity were observed in the PEC, in response to the treatment with 5 μM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.     

Expression of Masu Salmon Δ5-Desaturase-Like Gene Elevated EPA and DHA Biosynthesis in Zebrafish

Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Δ5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Δ5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Δ5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.     

Grouping Newly Isolated Docosahexaenoic Acid-Producing Thraustochytrids Based on Their Polyunsaturated Fatty Acid Profiles and Comparative Analysis of 18S rRNA Genes

Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.     

Dietary eicosapentaenoic acid and docosahexaenoic acid are linearly retained by common insect crop pests (cabbage looper and bertha armyworm) and alter insect biomass

The long-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are prevalent in aquatic ecosystems and are not part of the natural diet of herbivorous, terrestrial insects, which generally consume alpha-linolenic acid (ALA) and linoleic acid (LNA). However, recent advances in genetic engineering have lead to the development of terrestrial crops that express the novel traits of EPA and DHA production. In the present study, we examine the effects of dietary EPA and DHA on the growth, development and fatty acid content of two crop pest insects: bertha armyworm and cabbage looper. Five experimental diets were formulated to include increasing amounts of pure EPA and DHA (in relation to the total diet lipid level), according to the ratios (EPA + DHA relative to a vegetable oil containing ALA and LNA): 0 (control), 0.25 : 0.75 (lowest), 0.5 : 0.5 (low), 0.75 : 0.25 (medium) and 1 : 0 (high). Dietary EPA and DHA had significant effects on development time, mass and fatty acid content in both species. Dietary treatment (interactive with time) had a significant effect on individual mass of both insects, indicating that, over time, EPA and DHA impacted growth. However, insect mass, development and morphology results are not linearly related with increasing dietary EPA and DHA. Both species retained EPA and DHA in adult form, and the body content of EPA and DHA was significantly, positively correlated with EPA and DHA diet treatments in both the bertha armyworm (r² = 91.3%) and cabbage looper (r² = 75.8%). Dietary EPA and DHA could have fitness consequences for these organisms and could be nutritionally transferred to higher consumers.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs

Studies suggested that in human adults, linoleic acid (LA) inhibits the biosynthesis of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), but their effects in growing subjects are largely unknown. We used growing pigs as a model to investigate whether high LA intake affects the conversion of n-3 LC-PUFA by determining fatty acid composition and mRNA levels of Δ5- and Δ6 desaturase and elongase 2 and -5 in liver and brain. In a 2 × 2 factorial arrangement, 32 gilts from eight litters were assigned to one of the four dietary treatments, varying in LA and α-linolenic acid (ALA) intakes. Low ALA and LA intakes were 0.15 and 1.31, and high ALA and LA intakes were 1.48 and 2.65 g/kg BW0.75 per day, respectively. LA intake increased arachidonic acid (ARA) in liver. ALA intake increased eicosapentaenoic acid (EPA) concentrations, but decreased docosahexaenoic acid (DHA) (all P < 0.01) in liver. Competition between the n-3 and n-6 LC-PUFA biosynthetic pathways was evidenced by reductions of ARA (>40%) at high ALA intakes. Concentration of EPA (>35%) and DHA (>20%) was decreased by high LA intake (all P < 0.001). Liver mRNA levels of Δ5- and Δ6 desaturase were increased by LA, and that of elongase 2 by both ALA and LA intakes. In contrast, brain DHA was virtually unaffected by dietary LA and ALA. Generally, dietary LA inhibited the biosynthesis of n-3 LC-PUFA in liver. ALA strongly affects the conversion of both hepatic n-3 and n-6 LC-PUFA. DHA levels in brain were irresponsive to these diets. Apart from Δ6 desaturase, elongase 2 may be a rate-limiting enzyme in the formation of DHA.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells.