植物中γ-亚麻酸合成关键酶——△6-脂肪酸脱氢酶

γ-亚麻酸作为人体必需的不饱和脂肪酸,对人体的激素调节及脂肪酸代谢发挥着重要的生理作用.△6-脂肪酸脱氢酶是多不饱和脂肪酸合成途径中的限速酶.本文介绍了不饱和脂肪酸γ-亚麻酸代谢途径中的关键酶△6-脂肪酸脱氢酶的结构功能与目前△6-脂肪酸脱氢酶的基因工程研究进展,并对其应用进行了展望,以期为利用基因工程手段生产γ-亚麻酸提供参考.     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰_CoA羧化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果。     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰－ＣｏＡ羟化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果     

植物油脂的保健与药用价值

植物的油脂与人类的生活密切相关 ,其中保健与药用价值就是一个很重要的方面。在植物油脂各脂肪酸的组成中 ,不饱和脂肪酸 (如油酸、亚油酸和亚麻酸等 )为人体所必需。这类脂肪酸可防治中老年心血管硬化 ,并减少癌症的发病率。在上述不饱和脂肪酸中 ,以油酸对人体最为有利。它是细胞膜的必要成分 ,并且有降低人体内血清胆固醇和甘油三脂的功能 ,也能软化血管并防止血栓的形成。亚油酸和亚麻酸在人体内都不能合成 ,必须由食物供给 ,它们也是许多不饱和脂肪酸合成的前体 ,由前者形成的前列腺素类化合物等一系列代谢重要物质 ,对维持人体健康、…     

茄科雷尔氏菌脂酰CoA脱饱和酶和环丙烷脂肪酸合成酶的鉴定

茄科雷尔氏菌(Ralstonia solanacearum)是一种危害严重的土传植物致病菌,其宿主范围广泛,在世界各地严重影响重要经济作物的生产.研究茄科雷尔氏菌的生理特性,探索其致病机理,有利于研发防治青枯病的技术与方法.脂肪酸是细菌细胞重要的组成物质,但是茄科雷尔氏菌脂肪酸合成的机制尚不清晰.本文以茄科雷尔氏菌GMI1000为材料,鉴定了该菌的脂酰Co A脱饱和酶和环丙烷脂肪酸合成酶,并分析了这两种酶在不饱和脂肪酸和环丙烷脂肪酸合成中的作用.结果显示,茄科雷尔氏菌RSc2450编码脂酰Co A脱饱和酶,参与其不饱和脂肪酸合成,但是该菌还存在其他不饱和脂肪酸合成途径.同时发现在茄科雷尔氏菌编码两个可能的环丙烷脂肪酸合成酶蛋白质中,仅有Cfa1(RSc0776)参与了该菌环丙烷脂肪酸的合成,并在低p H和高渗透压的耐受中起作用.该研究结果为深入研究茄科雷尔氏菌脂肪酸合成代谢特点及致病机理奠定了基础.     

脂肪酸脱饱和的应用进展

脂肪酸脱饱和是由脂肪酸脱饱和酶所催化的不饱和脂肪酸合成途径的关键步骤。脂肪酸脱饱和酶分为脂酰CoA脱饱和酶、脂酰ACP脱饱和酶和脂酰脂脱饱和酶等三类。近年来脂肪酸脱饱和遗传操作在植物抗寒育种、植物油基因工程、食品工程、微生物发酵工程和植物抗害育种等方面的应用研究均取得了相当进展。     

脂肪酸脱饱和的应用进展

脂肪酸脱饱和是由脂肪酸脱饱和酶所催化的不饱和脂肪酸合成途径的关键步骤。脂肪酸脱饱和酶分为脂酰CoA脱饱和酶、脂酰ACP脱饱和酶和脂酰脂脱饱和酶等三类。近年来脂肪酸脱饱和遗传操作在植物抗寒育种、植物油基因工程、食品工程、微生物发酵工程和植物抗害育种等方面的应用研究均取得了相当进展。     

植物脂肪酸调控基因工程研究

脂肪酸代谢是植物最重要的代谢途径之一,脂肪酸在人们日常生活及工业生产上有重要用途,作者阐明了植物脂肪酸代谢的基本途径,并对近些年来植物脂肪酸代谢的遗传调控及基因工程的研究进展做了较为详细的总结,同时对植物脂肪酸调控基因工程发展提出了展望。     

植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用

植物磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)是广泛存在的一种细胞质酶,催化磷酸烯醇式丙酮酸(PEP)和HCO3-生成草酰乙酸(OAA),后者可转化生成三羧酸循环的多种中间产物.PEPC在植物细胞中参与植物的光合碳同化等重要代谢途径,并且在不同组织中具有多种生理功能.PEPC同时也参与调控植物种子的营养物质合成与代谢过程,控制糖类物质流向脂肪酸合成或蛋白质合成途径.以下介绍了植物PEPC的种类、蛋白质结构特点及其在植物组织中的调控方式,并重点论述了PEPC在生物基因工程中的应用方面的进展,随着对其功能机制和应用研究的深入,将有助于植物PEPC在高产优质农作物育种、能源植物和工业微生物等的开发利用等方面得到更好的发展与应用.     

苜蓿中华根瘤菌desA基因功能的鉴定

为研究苜蓿中华根瘤菌脂肪酸脱饱和酶desA基因在不饱和脂肪酸合成、共生结瘤固氮以及应对逆境胁迫中的功能,为高效利用苜蓿中华根瘤菌提供理论依据,本文通过异体遗传互补和脂肪酸组成薄层层析,分析SmdesA编码蛋白是否具有脱饱和酶的活性并参与不饱和脂肪酸的合成,构建SmdesA的缺失突变株和互补菌株,比较各菌株在不同逆境胁迫条件下的生长速率以及回接宿主植物后与紫花苜蓿共生结瘤的能力.结果表明SmdesA不能互补大肠杆菌CY57中EcfabA的突变,但具有将饱和脂肪酸脱饱和形成不饱和的棕榈油酸和十八碳烯酸的能力.另外,SmdesA缺失突变对苜蓿中华根瘤菌的脂肪酸组成影响不大,但会显著影响低温和高盐条件下菌株的生长速率以及与紫花苜蓿共生结瘤的能力.我们推测,SmdesA参与的脱饱和途径可能是苜蓿中华根瘤菌不饱和脂肪酸合成的补偿途径,其编码的蛋白DesA不是不饱和脂肪酸合成的关键酶,但在应对逆境胁迫和共生结瘤中具有重要的生物学功能.     

ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.     

植物脂肪酸去饱和酶及其编码基因研究进展

脂肪酸去饱和酶是催化脂肪酸链特定位置形成双键和产生不饱和脂肪酸的酶类。植物脂肪酸去饱和酶主要有5种(FAD2、FAD3、FAD6、FAD7和FAD8), 可分为ω-3型 (FAD2、FAD6)和ω-6型(FAD3、FAD7、FAD8)两大类。其编码基因(FAD2、FAD3、FAD6、FAD7和FAD8)在植物中一般有多个拷贝。同种基因在不同植物中拷贝数不同, 同一植物中相同基因的不同拷贝间在序列特征、表达调控和功能等方面也存在显著差异。本文根据国内外对脂肪酸去饱和酶基因及编码产物的研究现状, 分别从它们的分类、拷贝数、结构、作用机制、表达调控等方面的研究进展进行了详细的分类阐述。     

Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases.     

A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily.

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta12-acetylenase. The gene encoding this protein, together with a Delta6-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta5-, Delta6-fatty-acid desaturases and Delta8-sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b5-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Delta6-desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta9-unsaturated C16- and C18-fatty acids by inserting an additional Delta6cis-double bond. One of these transgenic yeast clones was also able to introduce a Delta6-triple bond into gamma-linolenic and stearidonic acid. This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Delta6-acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Delta6cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta6cis-double bond to a Delta6-triple bond.     

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.     

绿色巴夫藻脂肪酸去饱和酶的克隆和初步研究

在高等植物中,Δ9 脂肪酸去饱和酶引入第一个双键到饱和的脂肪酸链中,导致单不饱和脂肪酸的形成。我们通过RT-PCR、RNA ligase mediated RACE (RLM-RACE) and Overlap-PCR方法从海洋微藻绿色巴夫藻中克隆到一个命名为PvfadA的脂肪酸去饱和酶候选基因。通过将PvfadA基因在大肠杆菌表达系统中成功表达,PvFadA可以特异性地将C18:0脂肪酸转变成C18:1脂肪酸。PvFadA的氨基酸序列中存在一个存在于acyl-ACP去饱和酶的特异性金属离子结合区段(D/E)X2HX-100(D/E)X2H。通过同源模建PvFadA的3D结构显示,其包含了11个α螺旋,其中α3、α4、α6和α7组成了一个4个螺旋桶的核心结构,预测其可能是酶的活性中心。PvFadA的3D结构类似于蓖麻和结核分枝杆菌H37Rv的acyl-ACP去饱和酶。     

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.     

Fatty acid desaturation and the regulation of adiposity in Caenorhabditis elegans

Monounsaturated fatty acids are essential components of membrane and storage lipids. Their synthesis depends on the conversion of saturated fatty acids to unsaturated fatty acids by Delta9 desaturases. Caenorhabditis elegans has three Delta9 desaturases encoded by the genes fat-5, fat-6, and fat-7. We generated nematodes that display a range of altered fatty acid compositions by constructing double-mutant strains that combine mutations in fat-5, fat-6, and fat-7. All three double-mutant combinations have reduced survival at low temperatures. The fat-5;fat-6 double mutants display relatively subtle fatty acid composition alterations under standard conditions, but extreme fatty acid composition changes and reduced survival in the absence of food. The strain with the most severe defect in the production of unsaturated fatty acids, fat-6;fat-7, exhibits slow growth and reduced fertility. Strikingly, the fat-6;fat-7 double-mutant animals have decreased fat stores and increased expression of genes involved in fatty acid oxidation. We conclude that the Delta9 desaturases, in addition to synthesizing unsaturated fatty acids for properly functioning membranes, play key roles in lipid partitioning and in the regulation of fat storage.     

The role of cytochrome b5 fusion desaturases in the synthesis of polyunsaturated fatty acids

The biosynthetic pathway of polyunsaturated fatty acids (PUFAs) has been the subject of much interest over the last few years. Significant progress has been made in the identification of the enzymes required for PUFA synthesis; in particular, the fatty acid desaturases which are central to this pathway have now all been identified. These "front-end" desaturases are all members of the cytochrome b(5) fusion desaturase superfamily, since they contain an N-terminal domain that is orthologous to the microsomal cytochrome b(5). Examination of the primary sequence relationships between the various PUFA-specific cytochrome b(5) fusion desaturases and related fusion enzymes allows inferences regarding the evolution of this important enzyme class. More importantly, this knowledge helps underpin our understanding of polyunsaturated fatty acid biosynthesis.