白藜芦醇合酶的研究进展

白藜芦醇是一种重要的植物抗毒素,具有多种医疗保健作用,因此其应用前景非常广泛,已引起多方关注。白藜芦醇合酶是白藜芦醇生物合成途径中的关键酶之一,它催化1分子4—香豆酰辅酶A和3分子丙二酰辅酶A反应合成白藜芦醇,它是白藜芦醇生物合成中惟一必需的酶,关于它的研究已广泛开展起来。本文综述了白藜芦醇的药理活性、白藜芦醇合酶的酶学性质、诱导途径和机制以及分子生物学方面的研究进展。     

RS基因的植物表达载体和酵母表达载体构建

白藜芦醇合酶(RS)是Res生物合成的关键酶之一,它催化1分子4-香豆酰辅酶A和3分子丙二酰辅酶A反应合成Res.以花生中克隆的RS基因为基础,成功构建了RS基因的以Ubi为启动子的单子叶植物表达栽体pBIL-RS,为以后的基因工程遗传转化果蔗和其他单子叶植物改良其品质提供条件.同时构建了酵母表达载体pVT102U-RS,为下一步研究真核表达蛋白的生物活性提供条件,并为利用酵母生产Res提供了可能.     

异源表达CiRS基因通过生成白藜芦醇增强拟南芥的抗氧化能力

白藜芦醇是一种具有多种医疗保健作用的植物芪类次生代谢产物,在农业、医药、食品和化妆品等领域受到广泛的关注。白藜芦醇合酶是白藜芦醇生物合成中唯一必需的关键酶,决定植物体内白藜芦醇的合成。将中间锦鸡儿中克隆到的CiRS基因(Gen Bank登录号MF678590)转入野生型拟南芥,实验结果显示:野生型的总黄酮含量明显高于转基因株系。HPLC测得转基因拟南芥中有白藜芦醇的生成,并且含量最高达335μg/g FW。紫外照射处理后转基因植物中丙二醛的积累量明显少于野生型。转基因植物提取物DPPH自由基清除能力均高于野生型。这些结果表明,中间锦鸡儿CiRS基因异源表达后利用与黄酮类物质的共同底物合成了白藜芦醇,使得转基因植物的抗氧化性增强。     

花生白藜芦醇合酶基因的克隆与生物信息学分析

利用Protparam、iPSORT prediction、ProtScale、SOPMA、Swiss-Modeling和Scan Prosite等生物信息学工具分别对其理化性质、信号肽、疏水性、亲水性、二级结构和三级结构进行分析。以花生粤油45总DNA和总RNA为模板,采用PCR和RT-PCR技术克隆花生白藜芦醇合酶基因的DNA和cDNA序列,并利用SWISS-PROT、DNAMAN等生物信息学工具对其基因和蛋白质序列进行了分析。测序结果显示,该基因的DNA和cDNA序列长度分别为1 498 bp和1 251 bp,cDNA序列具有完整的开放性阅读框,编码389个氨基酸的多肽。该白藜芦醇合酶氨基酸368-378位点上存在芪合酶家族的特征位点GVLFGFGPGLT。同源性分析表明,其碱基序列与已报道的花生白藜芦醇合酶基因的一致性为99%,其氨基酸序列与已报道的花生白藜芦醇合酶氨基酸序列的一致性为100%。     

虎杖苯亚甲基丙酮合酶PcPKS2的表达纯化和晶体生长

植物类型Ⅲ聚酮合酶超家族(PKSs),又称查尔酮合酶(Chalcone synthase,CHS)超家族,催化合成多种植物次生代谢产物的分子骨架。苯亚甲基丙酮合酶(Benzalacetone synthase,BAS)催化4-香豆酰辅酶A与丙二酰辅酶A通过一步脱羧缩合反应生成苯亚甲基丙酮,是一系列具有重要生物学活性苯丁烷类化合物及其衍生物的前体化合物。前期工作从虎杖中分离出苯亚甲基丙酮合酶BAS(PcPKS2)和1个具有CHS和BAS活性的双功能酶(PcPKS1)。两者与超家族其他成员序列经比较,在包括门卫氨基酸Phe215和Phe265在内的重要氨基酸序列存在一定差异。已有蛋白晶体学研究结果表明,PKSs家族不同成员的功能多样性来自于酶催化位点的非常微小的构象变化。为了能够从结构上比较PcPKS2和Pc PKS1双功能酶活性差异可能产生的机制,以确定其高效BAS活性的分子机理,研究利用了大肠杆菌原核表达系统过量表达了C-端融合有His6标签的重组蛋白,经纯化得到了高纯度蛋白。经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究、了解虎杖聚酮类化合物生物合成机制和该类酶在基因工程中的应用提供了基础。     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotiana tabacum L.)中白藜芦醇产物的含量。MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合。芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.) Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SW强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草。Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物。与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍。MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础。     

产姜黄素大肠杆菌工程菌的构建

姜黄素是姜科植物的特征性成分,具有重要的药理活性.文中利用姜黄素生物合成关键酶β-酮酰辅酶A合酶(Diketide-CoA synthase,DCS)基因和姜黄素合酶(Curcumin synthase,CURS)基因构建非天然融合基因DCS::CURS,并将其与4-香豆酰辅酶A连接酶(4-coumarate coen...     

基于前体供给途径遗传改造提高褐黄孢链霉菌纳他霉素的产量

纳他霉素(natamycin)是一种高效、广谱、安全的抗真菌剂,广泛应用于食品防腐与医药领域。纳他霉素可由多种链霉菌发酵产生。它是以乙酰辅酶A、丙二酰辅酶A及甲基丙二酰辅酶A为前体经Ⅰ型聚酮合酶(polyketide synthase,PKS)催化合成的多烯大环内酯类化合物。本研究以纳他霉素产生菌——褐黄孢链霉菌为研究材料,分别对不同前体分子供给途径中的关键酶进行过表达,并确定影响纳他霉素产量的关键前体供给途径。研究结果发现：通过过表达乙酰辅酶A合成酶(acetyl-CoA synthase,ACS)加强乙酰辅酶A合成途径,以及通过过表达甲基丙二酰辅酶A变位酶(methylmalonyl-CoA mutase,MCM)加强甲基丙二酰辅酶A合成途径,重组菌株纳他霉素产量分别比野生型菌株提高了44.19%和20.51%。共过表达ACS和MCM,重组菌株纳他霉素产量获得进一步提升(达1123.34mg/L),比野生型菌株提高了66.29%。上述发现为通过前体代谢工程的策略构建纳他霉素工业高产菌株提供了参考,也为其他聚酮类天然产物高产工程菌株的构建提供了借鉴。     

前体代谢工程提高红霉素产量的研究进展

红霉素是十四元大环内酯类抗生素,具有广泛的医药价值和巨大的新药开发潜力。红霉素的主要成分红霉素A由丙酰辅酶A和甲基丙二酰辅酶A作为前体通过聚酮合酶合成大环内酯骨架,再经羟基化、糖基化、甲基化等一系列修饰合成。根据红霉素A的生物合成路线,我们从前体喂养途径、糖基化和甲基化优化等方面,简要综述近年来利用前体代谢工程手段提高红霉素产量的研究进展。     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

Resveratrol inhibits insulin responses in a SirT1-independent pathway

Resveratrol mimics calorie restriction to extend lifespan of Caenorhabditis elegans, yeast and Drosophila, possibly through activation of Sir2 (silent information regulator 2), a NAD+-dependent histone deacetylase. In the present study, resveratrol is shown to inhibit the insulin signalling pathway in several cell lines and rat primary hepatocytes in addition to its broad-spectrum inhibition of several signalling pathways. Resveratrol effectively inhibits insulin-induced Akt and MAPK (mitogen-activated protein kinase) activation mainly through disruption of the interactions between insulin receptor substrates and its downstream binding proteins including p85 regulatory subunit of phosphoinositide 3-kinase and Grb2 (growth factor receptor-bound protein 2). The inhibitory effect of resveratrol on insulin signalling is also demonstrated at mRNA level, where resveratrol reverses insulin effects on phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, fatty acid synthase and glucokinase. In addition, RNA interference experiment shows that the inhibitory effect of resveratrol on insulin signalling pathway is not weakened in cells with reduced expression of SirT1, the mammalian counterpart of Sir2. These observations raise the possibility that resveratrol may additionally modulate lifespan through inhibition of insulin signalling pathway, independently of its activation of SirT1 histone deacetylase. Furthermore, the present study may help to explain a wide range of biological effects of resveratrol, and provides further insight into the molecular basis of calorie restriction.     

Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses

Scavenging or quenching of the reactive oxygen species (ROS) involved in oxidative stress has been the subject of many recent studies. Resveratrol, found in various natural food products, has been linked to decreased coronary artery disease and preventing cancer development. The present study measured the effect of resveratrol on several different systems involving the hydroxyl, superoxide, metal/enzymatic-induced, and cellular generated radicals. The rate constant for reaction of resveratrol with the hydroxyl radical was determined, and resveratrol was found to be an effective scavenger of hydroxyl, superoxide, and metal-induced radicals as well as showing antioxidant abilities in cells producing ROS. Resveratrol exhibits a protective effect against lipid peroxidation in cell membranes and DNA damage caused by ROS. Resveratrol was also found to have a significant inhibitory effect on the NF-kappaB signaling pathway after cellular exposure to metal-induced radicals. It was concluded that resveratrol in foods plays an important antioxidant role.     

白藜芦醇合成酶基因在基因工程中的应用及功能研究进展

白藜芦醇合成酶(Resveratrol synthase,RS)是白藜芦醇(Resveratrol,Res)合成途径中的关键酶。以往研究报道,RS基因已在多种植物和微生物中进行了转化和表达,并在植物的代谢及调控等方面发挥生物学作用。文中主要围绕RS基因对植物的转化,及异源表达后植物体内代谢产物的变化,转RS基因对植物抗病原菌活性、抗自由基活性和生长发育的影响,以及利用RS基因在微生物中生产Res的相关进展进行了综述。并对RS基因在生物工程方面的应用前景进行展望。     

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.     

Production of a major stilbene phytoalexin, resveratrol in peanut (Arachis hypogaea) and peanut products: a mini review

As a major stilbene phytoalexin, resveratrol is produced or elicited in several plant species as a part of defense systems protecting plants against diseases. Resveratrol can be present in both the trans- and cis-isomeric forms, and only the trans-form increases the life expectancy and lowers the risk of cardiovascular diseases as the most bioactive form. In addition to the usages for diet and industry, peanut plant (Arachis hypogaea) and peanuts are getting higher attention due to their containment of resveratrol in the kernels and other parts of peanut plant, such as leaves, roots, and peanut shell. Recently, natural resveratrol derived from peanuts has also become a promising nutraceutical agent, promoting human health. Resveratrol has also been detected in peanut products including peanut butters, roasted peanuts, and boiled peanuts. Although, smaller and immature peanuts contain higher levels of resveratrol than mature peanuts, resveratrol in peanuts can also be preserved by cooking or manufacturing processes. Moreover, the amount of resveratrol in peanut plants and peanuts has been found to increase by external stimuli including microbial infection, wounding, UV light irradiation, ultrasonication, yeast extract treatment and by plant stress hormones. In addition, molecular level analysis has confirmed that four resveratrol synthase (RS) genes (RS1, RS2, RS3 and RS4) which catalyze synthesis of resveratrol have been identified in peanuts, and up-regulation of the genes is positively correlated to the increased contents of resveratrol. In this review, we summarize the natural biosynthesis of resveratrol in peanuts and peanut plants, as well as the occurrence of this natural phytoalexin in various peanut products. A brief knowledge on the biosynthetic pathway of resveratrol synthesis has been described. This review also deals on highlighting the effect of various external stimuli (biotic and abiotic stresses) in order to achieve the maximum induction and/or elicitation of resveratrol in peanuts and peanut plants.