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| A second problem with E. coli is the limited supply of complementary NADPH:cytochrome P450 reductase that is also essential for the correct function of reconstituted plant cytochrome P450 enzymes. Yeast does have endogenous microsomal cytochrome P450 enzymes and energy supporting systems, what is a major advantage for this host system.The taxadiene synthase encoding gene has also been expressed in A. thaliana. Constitutive expression of the gene led to taxadiene accumulation, but the A. thaliana plants showed growth retardation and decreased levels of photosynthetic pigment. The negative effects may have been caused by the toxicity of taxadiene, but more likely they are a result of the disturbance of the endogenous geranylgeranyldiphosphate pool. The use of an inducible expression system resulted in an increase of taxadiene accumulation. These findings clearly show that only the expression of heterologous genes result in the production of the desired compound, but the influence on the metabolic network has to be taken into account as well.Carotenoids are tetraterpenoids (C40 compounds) and produced in many plants and microorganisms. Their main biological function is the protection against oxidative damage and some are used as warning colours in plant defense system. The commercial interest for carotenoids can be explained mainly by their use as colorant, nutraceutical, or antioxidant in food and cosmetics. Next to that, it has been suggested that carotenoids could possibly play an important role as antic¬arcinogenic drug and in the prevention of chronic diseases. The carotenoid b-carotene is the primary source of Vitamin A in the human diet. The biosynthesis of carotenoids starts with the tail-to-tail coupling of two molecules of the general precursor GGDP by phytoene synthase (CrtB) resulting in the colorless carotenoid phytoene. Desaturation reactions inserting four additional double bonds in the molecule give eventually lycopene, the main carotenoid in tomato fruit, from which different cyclic and acyclic structures can be synthesized depending on the producing organism. Lycopene cyclase (CrtY) catalyses the cyclization at both ends of the lycopene molecule, resulting in two b-rings at the molecule b-carotene. Several other enzymes involved in the carotenoid biosyntheses have been identified, responsible not only for cyclization, but for glycosylation and diverse oxygenations as well.More than 600 different naturally occuring carotenoids have been identified so far. The three main carotenoids b-carotene, asthaxantin, and lycopene are produced by chemical synthesis and fermentation for commercial purposes. However, for carotenoids combinatorial biosynthesis in microorganisms is also described. Several carotenoid producing plants have been genetically modified to increase the production of the desired compounds. This review does not describe this research topic in detail, but the use of transgenic medicinal plants of Lycopersicum esculentem, Daucus carota, Solanum tuberosum, and Brassica napus has been reported. To overcome the problems with Vitamin A deficiencies in the third world, the biosynthetic pathway to b¬carotene engineered in rice (Oryza sativa) has led to the production of Golden Rice providing b-carotene, also referred to as pro-Vitamin A. Here we focus on the use of microorganisms for the production of carotenoids.The production of carotenoids by fermentation of carotenoid producing microorganisms such as Xanthophyllomyces den¬drorhous, Haematococcus pluvialis, and Blakeslea trispora has been investigated. X. dendrorhous produces 200–400 mg g~1 astaxanthin (85% of total carotenoid content). Engineering of X. dendrorhous by random mutagenesis led to an increase of 1.5–9 fold of the astaxanthin production inmutant strains. As a disadvantage of this approach growth inhibition and a decrease of biomass have been observed. More sophisticated recombinant DNA techniques introducing multi¬ple copies of genes encoding a bifunctional phytoene synthase/ lycopene cyclase and a phytoene desaturase also showed an increase in carotenoid production, but unexpectedly mostly other carotenoid structures than the desired astaxanthin (reviewed by Visser et al., 2003). Apparently, the hydroxylating enzyme became limited by overexpressing the mentioned enzymes. Several groups used gene clusters of Erwinia sp. for the expression in other hosts. In the last years several non¬carotenoid producing organisms have been explored for the production of carotenoids. This heterologous production is dependent on efficient expression systems for the carotenoid gene clusters, but increasing the supply of precursors in the host organisms is of importance as well. The yeasts Candida utilis and S. cereviseae have been engineered for the production of lycopene, b-carotene, and astaxantin. The prokaryote E. coli is most elaborated as a heterologous host, because most of the genes were already expressed in the strain for functional analysis. An overview of the heterologous expression of carotenoid gene clusters in the three mentioned non-carotenogenic hosts is described by The production of carotenoids in a host requires the biosynthesis of the intermediate GGDP. E. coli produces the C15 precursor FDP for endogenous terpenoid molecules. The extension of the prenyl chain to C20 has been performed by the expression of the CrtE gene encoding geranygeranyl dipho¬sphate synthase from Erwinia sp.. This prenyltransferase catalyses the production of GGDP from FDP. The GGDP synthase encoding gene gps from Archaeoglobus fulgidis has been expressed as well. Expression of this gene is more efficient, because the enzyme catalyses the three chain elongation reactions starting from the C5 precursors to the C20 molecule.One way to increase the heterologous production is to increase the pool of precursors in the host. Overexpression of several genes upstream in the isoprenoid biosynthesis resulted in the identification and overcome of bottlenecks in this pathway. Where the expression of a carotenoid gene cluster in C. utilis resulted in a lycopene production of 1.1 mg g~1 (dry weight) of cells, the overexpression of the catalytic domain of the HMG-CoA enzyme, involved in the isoprenoid biosynthesis via the mevalonate pathway, resulted in a 4-fold increase. Following disruption of the ergosterol biosynthetic gene ERG9 encoding squalene synthase yielded even more lycopene (7.8 mg g~1 (dry weight) of cells).To increase the isoprenoid flux in E. coli several genes of the DOXP pathway have been overexpressed. This resulted in a maximum increase of 10 times of the total carotenoid produc¬tion. Overexpression of genes encoding enzymes involved in a biosynthetic pathway is not always the solution for higher production levels, because they often cause an imbalance in the metabolic system of a host cell. Regulation of the supply of precursors and expression levels can contribute to the heterologous biosynthesis systems as well. The negative effects of overexpressing a rate limiting protein has been demonstrated for the deoxyxylulose phosphate synthase gene (dxs). The use of a multicopy plasmid containing a tac promoter resulted in a decrease of growth and lycopene production when expression was induced by IPTG where the dxs gene constructed on a low copy plasmid did not show these negative effects. Instead of plasmids the strong bacteriophage T5 promoter has been used to replace native promoters in E. coli. As a consequence the increased expression of isoprenoid genes led to improved production of lycopene (6 mg g~1 of dry cell weight) in E. coli. This production yield is comparable to the levels produced by carotenoid producing microorganims. Another approach to regulate the metabolic flux towards specific carotenoids has been observed by using a construct containing mRNA stability control elements. Varia¬tion of the mRNA stability modulated the flux of carotenoid production 300 fold towards b-carotene relative to lycopene.The balance of the starting precursors of the DOXP pathway has been investigated by Farmer and Liao. Over¬expression of several central metabolic genes redirected the flux of pyruvate towards glyceraldehyde 3-phosphate, resulting in an increase of lycopene in the heterologous E. coli strain. The same group also tried to design a controlled expression system for limiting enzymatic steps using an artificial intracellular loop.Since most carotenoid genes of different origin can function together in a host, combining several enzymatic combinations led to the production of new carotenoid structures not isolated from nature before.The use of host cells gives the opportunity to use directed evolution techniques for the modification of enzymes as well. Schmidt-Dannert et al. shuffled phytoene synthases of different bacterial species, which has resulted in a fully conjugated carotenoid containing six instead of four double bonds. The combination with shuffled lycopene synthases has shown production of the monocyclic carotenoid torulene. Extension of these pathways with other carotenoid modifying enzymes led to the production of novel structures in E. coli. Directed evolution has been used to create carotenoid-like molecules with different amounts of carbon atoms (C30, C35, C45 and C50) as well.Out of the group of terpenoids, the carotenoids have been most investigated in the production by naturally non-producing microorganisms and the production of new structures by combinatorial biosynthesis strategies. In contrast to the commercial interest, the pharmaceutical relevance of these compounds seems not to be of high importance at the moment. However, the knowledge out of this work can be applied for the heterologous production of other valuable terpenoid drugs like the mentioned artemisinin or paclitaxel. Although the avail¬ability of carotenoid gene clusters and promiscuity of the enzymes involved in the carotenoid biosynthesis are not present for structures of other terpenoids, the progress made, especially in engineering the upstream pathway creating a higher flux ofgeneral isoprenoid precursors, can be useful for all terpenoid structures as counts for the directed evolution techniques as well. |
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waiting113
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2楼2008-02-13 11:53:42
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| 第二个问题与大肠杆菌是供应有限的互补性和NADPH ：细胞色素P450还原酶，那也是必不可少的正确功能重组植物细胞色素P450酶。酵母是否有内源性微粒体细胞色素P450酶和能源的支撑体系，什么是一大优势，为这主系统紫杉合酶编码基因，也有人表示，在拟南芥。构基因的表达，导致紫杉积累，但拟南芥植物表明，生长迟缓，并减少各级光合色素。负面效应可能已经造成的毒性紫杉，但更可能是一个结果，干扰了内源geranylgeranyldiphosphate池。使用一种诱导表达系统，导致增加了紫杉积累。这些结果清楚地表明，只有表达外源基因，结果在生产理想的复合型，但影响，对代谢网络必须考虑到作为well.carotenoids是tetraterpenoids （ c40化合物） ，并制作了许多植物和微生物。其主要生物学功能是保护，防止氧化损伤的，有些是用来作为预警颜色在植物防御系统。商业利益为类胡萝卜素可以解释，主要是利用他们作为着色剂， Nutraceutical公司，或抗氧化剂，在食品和化妆品。旁边，有意见认为，类胡萝卜素可能扮演一个重要的角色，作为antic行政长官arcinogenic药物和预防慢性疾病。类胡萝卜素的B胡萝卜素的主要来源是维生素A在人的饮食习惯。合成类胡萝卜素从尾部到尾耦合的两个分子的一般前兆ggdp由phytoene合酶（ crtb ） ，导致在无色类胡萝卜素phytoene 。饱和反应插入增设4个双键的分子，最终让番茄红素，类胡萝卜素主要在番茄果实中，从不同的循环和非循环结构，可以合成依赖于生产的有机体。番茄红素环化酶（ crty ）催化环在两端的番茄红素分子，造成两架B型环在分子的B胡萝卜素。其他几个酶参与类胡萝卜素biosyntheses已经确定，不仅负责环，但对于糖基化和多样化oxygenations作为well.more超过600个不同的自然发生的类胡萝卜素已查明至今。三个主要的类胡萝卜素的B胡萝卜素， asthaxantin ，番茄红素是由化学合成和发酵作商业用途。然而，对类胡萝卜素的组合生物合成中的微生物是也进行了描述。几类胡萝卜素的生产厂房已经基因改造，以增加生产所期望的化合物。这项检讨工作，并不说明这一研究课题细节，但使用转基因药用植物的lycopersicum esculentem ，胡萝卜，马铃薯，甘蓝型油菜已报警。去克服困难，与维生素A缺乏症在第三世界，生物合成途径向B ，你胡萝卜素工程，在水稻（ Oryza sativa ） ，导致生产的黄金稻米提供的B胡萝卜素，也称为亲维生素A这里我们侧重于利用微生物生产carotenoids.the生产类胡萝卜素的发酵生产类胡萝卜素生产的微生物如xanthophyllomyces书斋，你drorhous ，雨生红球藻，和三孢布拉霉菌警方已展开调查。十dendrorhous产生200-400毫克G 〜为一虾青素（ 85 ％的总类胡萝卜素含量） 。工程中的十大dendrorhous通过随机突变，导致增加了1.5-9倍的虾青素生产inmutant株。由于这种方法的缺点生长抑制和减少了生物质能已被观察到。更精密的DNA重组技术引进多，你重份的基因编码一个双功能phytoene合酶/番茄红素环化酶和phytoene脱氢酶也增加，这说明在类胡萝卜素生产，但没想到大部分其他类胡萝卜素的结构比预想虾青素（审查visser等人， 2003年） 。很显然， hydroxylating酶成为有限的，由过上述酶。几个小组利用基因簇的软腐病菌藻为表达在其他主机。在过去的几年中一些非行政长官产生类胡萝卜素的生物体进行了探讨，为生产类胡萝卜素。这种异种生产是依赖于高效表达系统，为类胡萝卜素的基因簇，但增加的供应量前体在宿主生物体，是具有重要意义的。该酵母菌念珠菌有用和嗜cereviseae已改造为生产番茄红素和B -胡萝卜素，虾青素。该原核生物大肠杆菌是最阐述了作为异种主机，因为大部分的基因，已经体现在应变功能分析。概述了异种表达的类胡萝卜素的基因簇在三个提及非carotenogenic主持人介绍所生产的类胡萝卜素在东道国需要合成的中间ggdp 。大肠杆菌产生c15前兆FDP的内源性萜类分子。扩建的异戊烯链c20已经演出表达了crte基因编码geranygeranyl dipho行政长官sphate合酶欧文sp ..这prenyltransferase催化生产ggdp从果糖。该ggdp合酶编码基因的全球定位系统由archaeoglobus fulgidis一直表现为好。表达这种基因是更有效率的，因为酶催化的三个链伸长反应，从碳五前兆向c20 molecule.one方式，以增加外源生产，是增加池体在东道国。过度的几个基因的上游，在类异戊二烯生物合成的结果是确定和克服的瓶颈，在这方面的门路。凡表达一种类胡萝卜素的基因簇在C有用导致了番茄红素的生产1.1毫克G 〜为1 （干重）的细胞，过度的催化区的保守区农委的酶，参与类异戊二烯生物合成经mevalonate通路，造成4倍的增幅。下列扰乱了麦角固醇的生物合成基因erg9编码鲨烯合酶的产生更是番茄红素（ 7.8毫克G 〜为1 （干重）的细胞） ，以增加类异戊二烯通量在大肠杆菌中的几个基因的doxp通路已过度。这就产生了一个最高增加了10倍，总胡萝卜素生产瑃ion 。过度表达的基因编码的酶在生物合成途径并不总是解决方案较高的生产水平，因为它们往往造成一种不平衡的新陈代谢系统的寄主细胞。规例的前体供应和表达水平能有助于推动异种生物系统，以及环境。负面影响过速度极限的蛋白质已被证明为deoxyxylulose磷酸合酶基因（ dxs ） 。使用一种multicopy质粒载有交谘会启动，造成减少的增长和生产的番茄红素时，表达IPTG诱导下dxs基因建造一个低拷贝质粒，并没有显示这些不利影响。而非质粒强烈噬菌体T5的启动子已被用来取代本土启动子在大肠杆菌中。作为一个后果，表达的增加，类异戊二烯基因导致改进生产番茄红素（ 6毫克G 〜为1干重） ，在大肠杆菌中。这一产量是相媲美的水平，所产生的类胡萝卜素生产microorganims 。另一种办法，以规管代谢通量对特定类胡萝卜素已观察到用建构含有mRNA稳定性控制要素。杂物瑃ion的mRNA稳定性调制通量的类胡萝卜素生产的300倍，实现的B胡萝卜素相对lycopene.the余额出发前体的doxp通路警方已展开调查，由农民与廖。过去，你的表达数个中央代谢酶基因重定向通量丙酮酸对甘油三磷酸，因而增加了番茄红素在异种大肠杆菌菌株。同一集团也试图设计一套控制系统中的表达限制酶法步骤，用人工胞loop.since大多数类胡萝卜素的基因，不同来源的功能，可以一起在一个主持人，结合多种酶的组合，导致生产新的类胡萝卜素的结构不是孤立的，从性质before.the利用宿主细胞给人的机会，利用定向进化技术，为改造酵素等。施密特- dannert等人。洗牌phytoene合成酶的细菌不同物种，这导致了在一个完全共轭类胡萝卜素含6个而不是4个双键。结合洗牌番茄红素合酶表现出生产的单环类胡萝卜素torulene 。扩大这些通路与其他类胡萝卜素修饰酶，导致生产的新型结构在大肠杆菌。定向进化已被用来制造类胡萝卜素分子与不同数目的碳原子（ c30 ， c35 ， c45和C50型）作为well.out该集团的萜类化合物，类胡萝卜素被调查的大部分在生产由自然非生产微生物和生产新的结构组合生物合成策略。相反，以商业利益，制药相关的这些化合物似乎没有予以高度重视，在时刻。然而，知识出这方面的工作可以用于异种生产其他有价值的萜类药物一样，提到青蒿素或紫杉醇。尽管是徒劳的，你有能力的类胡萝卜素的基因簇及滥交的酶参与了类胡萝卜素的合成是没有出席，为结构的其他萜类化合物，所取得的进展，特别是在工程上游通路创造更高的通量ofgeneral类异戊二烯的前体，可用于所有萜类结构作为计数为定向进化技术等。 |
3楼2008-02-13 13:41:24
4楼2008-02-13 14:10:43
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waiting113(金币+2,VIP+0):翻译的不错
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A second problem with E. coli is the limited supply of complementary NADPH:cytochrome P450 reductase that is also essential for the correct function of reconstituted plant cytochrome P450 enzymes. Yeast does have endogenous microsomal cytochrome P450 enzymes and energy supporting systems, what is a major advantage for this host system.The taxadiene synthase encoding gene has also been expressed in A. thaliana. Constitutive expression of the gene led to taxadiene accumulation, but the A. thaliana plants showed growth retardation and decreased levels of photosynthetic pigment. The negative effects may have been caused by the toxicity of taxadiene, but more likely they are a result of the disturbance of the endogenous geranylgeranyldiphosphate pool. The use of an inducible expression system resulted in an increase of taxadiene accumulation. These findings clearly show that only the expression of heterologous genes result in the production of the desired compound, but the influence on the metabolic network has to be taken into account as well. 有关大肠杆菌的第二个问题是：限制补充NADPH，细胞色素P450还原酶对于再造植物细胞色素P450酶的正确功能所必须的。酵母有内源性的微粒体和能量供给系统，这个系统对于宿主系统是个主要的有点。紫杉合成编码基因也在A。拟南芥中表达。基因的组成型表达是紫杉得意积累，但A。拟南芥植物表现出生长迟缓和光合色素水平减少。这种负面的作用可能是由于紫杉的毒性，但也更可能是内源？？牛龙牛儿基氯喹群紊乱的结果。使用诱导性表达最终导致紫杉积累增加。这些发现清楚的显示：仅仅异源表达一进可以产生希望得到的产物，但由于代谢的交联作用影响其他的也被积累。 |
5楼2008-02-13 20:45:51
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Carotenoids are tetraterpenoids (C40 compounds) and produced in many plants and microorganisms. Their main biological function is the protection against oxidative damage and some are used as warning colours in plant defense system. The commercial interest for carotenoids can be explained mainly by their use as colorant, nutraceutical, or antioxidant in food and cosmetics. 胡萝卜素是tetraterpenoids（C4混合物）许多植物和微生物均可产生，他们主要的生物学功能是防止氧化损伤和在植物保护系统中作为保护色。胡萝卜素的商业用途是作为染料、营养物货食物、化妆品的抗氧剂。 |
6楼2008-02-13 20:48:57
taijun_2008
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