Name: the logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin a produced through e. coli
Uploaded: 2013-01-13T14:00:00
Description: Watch this Scientific Journal Video about The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E.

作者感谢美国国立卫生研究院（AI074224和GM085323）和美国国家科学基金会（0712019和0924699）提供资金支持项目，致力于异源生物合成。

下面的文字是红霉素A抗生素的具体的功能，但该步骤被设计成一般适用于异源生物合成的其它天然产物的作为候选。 1。红霉素的遗传集群式转移<ol><li>设计PCR引物扩增的所有的相关的基因的红霉素A群集内的S.红霉素染色体。这一步是针对那些自然的候选产品，其基因序列已被确定。或者，所需的基因，可以合成（通过几个商业供应商选项）的优点，新合成的基因将?...

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异源生物合成中的关键步骤是在每个点的过程的三个程序：1）基因转移2）生物合成的重建，以及3）产品的分析中遇到。在任何阶段的问题，将破坏建立异源生物合成的最终目标。也许是最具挑战性的方面建立重组生物合成的过程，因为这是绝对必要的，让成功的分析。然而，重组是依赖于精心的设计和负责最终合成的遗传物质的转移。同样地，建立，灵敏的分析方法（如LC-MS）可以检测到小的?...

红霉素A是由革兰氏阳性土壤细菌糖多孢菌红霉素产生聚酮化合物的抗生素，和目前的生产已增量通过几十年的传统的诱变和筛选协议和最近通过过程优化方案1-6〜10 g / L的改善。诱变和筛选策略是常见的抗生素天然产品开发的困难，因此在培养和/或基因操纵本地生产主机，因为现成的抗菌活性或改进的的增长表型，以帮助选择。红霉素A的情况下，S.红霉素是一个缓慢的增长模式缺乏更直接的遗传操作技术（生物如大肠杆菌 ）的限制，因此，阻碍了生产的生物合成新的衍生产品的迅速改善。经确认的生产问题和解锁diversificati化合物，如红霉素A的可能性，研究界开始追求的理念异源生物合成（ 图1）7。这些努力的同时提供序列信息的红霉素A基因簇8-11。应当强调的是，序列复杂的天然产物的基因簇的数目已大大扩展12-16，提供的动力继续努力，在异源生物合成访问编码药用潜力。要做到这一点，异源的重建需要的新的主机满足的特定的生物合成途径的需要。E.大肠杆菌提供技术方便，一个广泛的分子生物学技术的跨越式组，代谢和制程工艺的产品发展战略。然而，相对于本地生产主机，E.大肠杆菌不表现出复杂的天然产物的生产相同的水平。因此，未知是否Ë。大肠杆菌可以作为一个可行的复杂的天然产物生物合成的异源的选择。然而，它被假定， 大肠杆菌大肠杆菌是一个理想的宿主生物体，可以实现异源生物合成。 有了这个共同的目标，最初的努力开始生产聚酮苷元6的deoxyerythronolide的B（6dEB）通过E.大肠杆菌 。但是，本机E.大肠杆菌代谢不能提供相当水平的丙酰辅酶A，和（2S） -甲基丙酰辅酶A需要支持6dEB生物合成的前体，也可以在新主机翻译后修改deoxyerythronolide的乙合酶（DEBS）酶。为了解决这些问题，由本地和外源酶的代谢途径是内置到E.大肠杆菌等，外生馈丙酸转化细胞内丙酰辅酶A，然后（2S） -甲基丙酰辅酶A，在工程完成这一途径，SFP基因放入第E染色体E.大肠杆菌 BL21（DE3）中，以产生一个新的菌株称为BAP1。的SFP酶是一个phosphopantetheinyl的的转移能够连接的4&#39;-phosphopantetheine上的辅助因子DEBS酶17,18。 DEBS基因（每个〜10 KB），然后放置在两个独立的可选诱导的T7启动子的表达载体。诱导后的温度（〜22℃）的一个关键调整后，DEBS基因协调表示内BAP1在启用状态中，能够产生6dEB 19。 追求全红霉素生物合成，然后开始使用类似的基因簇孢megalomicea或从S.的基因组成的混合途径红霉素，S.弗氏和S. venezuelae生产的中间体红霉素C和ð6 deoxyerythromycin，分别为20日至22日 。最近，我们扩展了这些努力，生产erythrom通过E.霉素A（红霉素）的临床相关的形式大肠杆菌 。在以前的工作中，我们的战略协调原来的20 S.红霉素的基因所需的聚酮化合物的生物合成，deoxysugar生物合成和附件，额外的剪裁，自电阻（ 图2）。总体而言，26（本地和外源）基因被设计为允许E.大肠杆菌产生红霉素A在4毫克/升23,24。这一结果建立了完整的生产一个复杂的聚酮的天然产物E.大肠杆菌和服务为基础，以利用这个新的生产选项，或寻求新的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> PCR仪</td><td> Eppendorf公司</td><td>的Mastercycler个人</td><td></td></tr><tr><td>二甲亚砜（DMSO） </td><td>费舍尔</td><td> BP231 </td><td></td></tr><tr><td>电穿孔</td><td> BioRad公司</td><td> Micropulser </td><td></td></tr><tr><td> IPTG </td><td>费舍尔</td><td> BP1620 </td><td></td></tr><tr><td>丙酸钠</td><td>西格玛</td><td> P1880 </td><td></td></tr><tr><td> L-阿拉伯糖</td><td>西格玛</td><td> A3256 </td><td></td></tr><tr><td>冷冻振荡器</td><td> Thermo Scientific的</td><td> MAXQ 4000 </td><td></td></tr><tr><td>微型离心</td><td> Ëppendorf </td><td>离心5415D </td><td></td></tr><tr><td> pGro7 </td><td>宝</td><td>伴侣质粒集（3340） </td><td></td></tr><tr><td> pET21，pET28，PCDF的二重奏-1 </td><td> EMD化学品</td><td> 69742-3，69864，71340 </td><td></td></tr><tr><td> LC-MS </td><td>美国应用生物系统公司</td><td> 3200 Q-陷阱</td><td></td></tr><tr><td>乙酸乙酯</td><td>西格玛</td><td> 270989 </td><td></td></tr><tr><td>甲醇</td><td>西格玛</td><td> 322415 </td><td></td></tr><tr><td>真空离心机</td><td> Eppendorf公司</td><td>集中5301 </td><td></td></tr><tr><td>旋转蒸发仪</td><td>步琪</td><td> R-200 </td><td></td></tr></tbody></table>

the logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin a produced through e. coli

复杂的天然产物的异源生产的是一种方法，旨在解决目前的局限性和未来的可能性。这是特别有用的那些化合物具有治疗价值，但不能充分地生产或将受益于生产一种改进形式的。实验过程可以细分成三个部分：1）基因转移; 2）异源重组; 3）产品分析。每个实验的成分是在不断优化，迎接挑战和预测的机会与这个新兴的方法。 异源生物合成的基因序列的识别珍贵的天然产品开始。此序列传送到一个​​异源宿主是复杂的生物合成途径的复杂性负责产物的形成。抗生素红霉素A是一个很好的例子。二十基因（最后的合成，共计50 KB）所需要的。此外，这些基因编码megasynthases的，域多酶〜300 kDa的大小。这种遗传材料必须被设计，并转移到大肠杆菌大肠杆菌重组生物合成。 PCR分离，操纵子建设，多cystronic的质粒，和电变换的使用将在下文中转移遗传聚类的红霉素A 至 E。 大肠杆菌 。 一旦转移，E.大肠杆菌细胞必须支持最终合成。这个过程也是充满挑战的巨大差异之间E.大肠杆菌和最原始的主机，负责复杂的天然产物的生成。该单元必须提供必要的支持生物合成的底物，并协调转移的基因簇表达活性酶。红霉素A，E 的情况下，在大肠杆菌细胞被设计，以提供两个前体（公关opionyl-CoA和（2S） - 甲基丙-CoA）的生物合成所需的。此外，基因序列的修改，质粒拷贝数，分子伴侣共表达，翻译后的酶修饰，和过程温度，也可以进行最后的红霉素A的形成。 最后，成功的生产必须进行评估。红霉素A的情况下，我们将介绍这两种方法。第一个是液相色谱 - 质谱（LC-MS），以确认和量化生产。也将红霉素A的生物活性的生物测定中， 枯草芽孢杆菌（Bacillus subtilis）的抗菌活性的测试是针对通过使用确认。评估分析，建立红霉素生物合成E.大肠杆菌 ，并设置为未来的工程努力，以改善或生产多样化和使用此方法的新的复杂的天然化合物的生产阶段。

这种方法所期望的结果是完全具有生物活性的天然产物生产的E.大肠杆菌异源宿主。这是最好的表示的LC-MS的结果，用于确认和量化生产（ 图6）和用于生物测定，以确认最后的活动（ 图7）的抗菌。在整体方案中的异源生物合成，这样的结果来定义成功。一旦完成，研究工作转向优化（无论是在细胞和工艺尺度）与分子工程。的最终目标包括原始化合物在异源系统?...

Watch this Scientific Journal Video about The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. 

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

通过异源生物合成红霉素A<em&gt; E.大肠杆菌</em包括下列实验步骤：1）基因转移; 2）异源重建;和3）产品分析。在生产的天然产物药物的使用，每个步骤进行说明的上下文中的动机，电势，和挑战<em&gt; E.大肠杆菌</em作为代理主机。

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过<em&gt; E.大肠杆菌</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Research

JoVE Journal

Biology

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Processing the Loblolly Pine PtGen2 cDNA Microarray

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

人类诺沃克病毒凸，域的生产<em&gt;电子。大肠杆菌</em&gt; X射线晶体学

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

诱导位点特异性复制遇阻<i&gt;电子。大肠杆菌</i&gt;用荧光阻遏操作员系统

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...