Name: identifying amino acid overproducers using rare-codon-rich markers
Uploaded: 2019-06-24T13:30:00
Description: Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

这项工作得到了国家自然科学基金(授权号21676026)、国家重点研发项目(批号2017YFD0201400)和中国博士后科学基金会(批号2017M620643)共同支持。加州大学洛杉矶分校(苏州)的工程得到了江苏省和苏州工业园区的内部资助。

1. 表达稀有富含柯顿标记基因的质粒构造
<ol><li>选择一个标记基因,其中包含目标氨基酸的相应数量的常用通子。 注:对于L-亮氨酸,卡那霉素抗基因kan R,其中含有29个亮氨酸科顿,其中27个是常见的科顿,用于构建选择系统13。gfp基因,包含17个普通科顿从19个亮子,或紫色蛋白质编码基因prancerpurple(ppg),其中含有14个亮氨酸常见的科顿,用于筛选系统(<s...

<ol>
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标记基因和选择或筛选培养基中的稀有科顿数量对于抑制稀有科顿修饰标记基因的蛋白质表达至关重要。如果野生型标记基因的蛋白质表达及其衍生物之间没有显著差异,增加稀有科顿的数量或使用营养有限的培养基可能会放大差异。然而,如果抑制作用太强,即使额外喂食相应的氨基酸,蛋白质表达也可能无法恢复。在这种情况下,标记基因中的稀有科子数量应减少,以减轻部分压力。微调选择或筛选?...

目前氨基酸的生产严重依赖发酵。然而,大多数氨基酸生产菌株的分度和产量低于全球氨基酸市场日益增长的需求,价值数十亿美元1,2。获得高性能氨基酸过度生产者是氨基酸产业升级的关键。
传统的识别氨基酸过度生产者的策略利用氨基酸和其模拟在蛋白质合成3,4之间的竞争。这些模拟物能够给识别相应氨基酸的tRNA充电,从而抑制肽链的增长,导致被抑制的生长或细胞死亡5。抵抗模拟应力的一种方法是增加细胞内氨基酸的浓度。富集氨基酸将超越有限tRNA的模拟,并确保功能蛋白的正确合成。因此,可以选择在模拟中存活的菌株,并且可能是相应氨基酸的过度生产者。
虽然证明在选择L-亮氨酸6等氨基酸的过度生产者方面是成功的,但基于模拟的策略存在严重缺陷。一个主要问题是来自诱变过程或自发突变的模拟阻力。具有电阻的应变可以通过阻塞、导出或降解模拟5来逃避选择。另一个担忧是模拟对其他细胞过程的毒性副作用7。因此,在模拟选择中幸存下来的菌株可能不是氨基酸过度生产者,而所需的过度生产者可能由于负面的副作用而被错误地消灭。
本文提出了一种基于科顿偏倚定律的新策略,以便准确、快速地识别氨基酸过度生产者。大多数氨基酸由多个核苷酸三联体编码,由宿主生物体8、9以不同的方式青睐。有些科顿很少在编码序列中使用,被称为稀有的科顿。它们转化为氨基酸依赖于携带相应氨基酸的干酪tRNA。然而,识别稀有科顿子的tRNA通常比普通科顿10、11的tRNA的丰度低得多。因此,这些稀有的tRNA不太可能在与其他异位接受器的竞赛中捕获游离氨基酸,当氨基酸的量有限时,稀有富含克顿序列的翻译开始减速甚至终止10.理论上,如果由于过度生产或额外喂食相应的氨基酸12而对同义的通用tRNA收费后,如果存在氨基酸过剩,翻译可以恢复。如果稀有的富含科顿的基因编码了选择或筛选标记,则表现出相应表型的菌株很容易被识别,并且很可能是靶向氨基酸的过度生产者。
应用上述策略建立氨基酸超标生产者的选型和筛选系统。选择系统使用抗生素抗性基因(例如kan R)作为标记,而筛选系统使用编码荧光(例如绿色荧光蛋白 [GFP]) 或色基因(例如 PrancerPurple)蛋白质的基因。通过用同义的稀有替代品替换目标氨基酸的常用通子的定义为号的标记基因,两个系统中的标记基因被修改。在突变库中,含有稀有的富含柯顿标记基因的菌株在适当的条件下被选择或筛选,并且靶向氨基酸的过度产生者很容易被识别。工作流程从构建稀有的富含柯顿标记基因系统开始,然后优化工作条件,然后对氨基酸过度生产者进行鉴定和验证。这种模拟独立策略基于蛋白质翻译中的教条,并已实际验证,能够准确、快速地识别氨基酸过度生产者。从理论上讲,它可以直接用于氨基酸与罕见的科顿和所有微生物。总之,当特定氨基酸无法使用适当的模拟时,或者当高误阳性率是主要问题时,基于稀有的节子策略将作为传统基于模拟的方法的有效替代方法。下面的协议使用亮氨酸稀有科顿来演示这一策略,以识别大肠杆菌L-亮氨酸过度生产者。

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo</td>
			<td>51101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure HiPure Plasmid MiniPrep Kit</td>
			<td>Transgen</td>
			<td>EM111-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>Transgen</td>
			<td>EG101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson assembly master mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane</td>
			<td>Thermo</td>
			<td>H3061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenyl isothiocyanate</td>
			<td>Sigma</td>
			<td>P1034</td>
			<td></td>
		</tr>
		<tr>
			<td>PrancerPurple CPB-37-441</td>
			<td>ATUM</td>
			<td>CPB-37-441</td>
			<td></td>
		</tr>
		<tr>
			<td>TransStar FastPfu Fly DNA polymerase</td>
			<td>Transgen</td>
			<td>AP231-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-high performance liquid chromatography</td>
			<td>Agilent</td>
			<td>1290 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild type C. glutamicum</td>
			<td>ATCC</td>
			<td>13032</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold E. coli competent cell</td>
			<td>Agilent</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX RRHD Eclipse Plus C18 column</td>
			<td>Agilent</td>
			<td>959759-902K</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying amino acid overproducers using rare-codon-rich markers

为了满足不断增长的氨基酸市场,需要高性能生产菌株。氨基酸过度生产者通常是通过利用氨基酸与其模拟物之间的竞争来识别的。然而,这种基于模拟的方法精度较低,特定氨基酸的正确模拟是有限的。在这里,我们提出了一个替代策略,利用稀有富含柯顿标记的氨基酸过度生产者进行准确、敏感和高通量筛选。这一策略的灵感来自于蛋白质翻译中的柯顿使用偏差现象,根据在编码DNA中出现的频率,将科顿分为普通或稀有。稀有科顿子的翻译取决于其相应的罕见转移RNA(tRNA),在饥饿的情况下,这种RNA不能被共生氨基酸完全充电。从理论上讲,如果向同义的通用等接受器充电后,氨基酸过剩,则可以对稀有tRNA收费。因此,通过喂食或细胞内过度生产相应的氨基酸,可以恢复由稀有科顿引起的缓速翻译。根据这一假设,通过用抗生素耐药性基因或基因中的同义词稀有替代品取代靶向氨基酸的常见子细胞,建立了用于识别氨基酸过度生产者的选择或筛选系统编码荧光或致色蛋白。我们表明,蛋白质的表达会因稀有的科顿而受到很大阻碍,蛋白质水平与氨基酸浓度呈正相关。利用这个系统,多种氨基酸的过产可以很容易地从突变库中筛选出来。这种基于稀有的基于科顿的策略只需要一个修饰基因,并且宿主比其他方法更不可能逃脱选择。它提供了一种获得氨基酸过度生产者的替代方法。

对于选择系统,与在合适的培养中培养野生型抗生素耐药性基因的菌株相比,应观察到携带稀有富柯顿抗生素耐药性基因的菌株的OD600的急剧减少。中等 (图 1a)。在相同的条件下,随着抗生素耐药性基因中稀有科顿数量的增加,细胞OD600的减少变得更加明显(图1a)。应该指出的是,对蛋白质表达的稀有科顿抑制大多发生?...

Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

本研究提出了一种替代策略,即使用稀有富含柯顿标记物的氨基酸过度生产者识别氨基酸,同时实现精度、灵敏度和高通量。

使用稀有的科顿富标记识别氨基酸过度生产者

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

这种方法为传统的模拟方法提供了获得氨基酸超生产器的高效替代方案。该技术通过同时提供精度、灵敏度和高吞吐量，比传统的基于模拟的方法更上一种。该方法为理解氨基酸生产强度和稀有龙子的转化提供了新的依据，从理论上可以应用于所有微生物。该协议主要依靠基本的分子实验操作，这些操作易于遵循，但可能需要多次试验，以实现应变应变的合适选择。该技术的视觉演示提供了对选择和筛选系统在识别氨基酸生产者方面的性能的直接了解。要开始此过程，请将矢量添加到标记片段中，在冰上，以一比一比七点五微升的装配混合到总体积十微升的摩尔比。在50摄氏度下孵育一小时。在 42 摄氏度下将装配产品的 5 微升转换为 50 微升组件单元，使用 30 秒。在37摄氏度下用卡塔博利特抑制介质恢复超级最佳肉汤中的细胞一小时。然后，在LB Agar介质上盘，在37摄氏度下孵育过夜。第二天，使用首选的商业套件来隔离质粒。首先，用一个微升质粒，携带野生型KanR，转化50微升的称职细胞。用含有KanR RC 29的质粒转换第二组称职细胞，所有白氨酸编码素被稀有的codon CTA所取代。像文本协议中概述的培养细胞和孵育细胞培养。接下来，将含有野生型选择标记基因的菌落分别转移到五毫升的五毫升稀释LB介质中，每毫升浓度为50微克。在 37 摄氏度的摇床中孵育样品，同时在 250 rpm 时摇动。然后，在定义的时间点将每个细胞培养物的 200 微升转移到 96 井板中。使用板读卡器测量 OD600。在含有卡那霉素的两组五毫升的 0.2 x LB 介质中接种含有 KanR RC 29 标记基因的污渍，带或不供应 L-leucine。将 L-leucine 添加到其中一个介质中，最终浓度为每升一克。在 37 摄氏度的摇床中孵育样品，在 250 rpm 时摇动，并在确定的时间点测量每个培养的 OD600。首先，用一种携带野生型GP或野生型PPG的质粒的微升来转化用于突变的父株的50微升。板和孵育的细胞培养，如文本协议中概述。接下来，将菌落单独转移到正确稀释的 LB 介质的五毫升。在 37 摄氏度的摇床中孵育样品，在 250 rpm 时摇动。对于荧光标记，在定义的时间点将 200 微升的细胞培养物转移到 96 个清晰、清晰的底部背板中。测量荧光中的OD600并计算荧光强度。对于色度标记，测量细胞培养物的颜色发展。执行如前所述的进料测定，测量荧光强度或定义时间点的颜色发展。首先，使用携带选择标记KanR RC 29或筛选标记GFP RC或PPG RC的质粒的一微升，转换突变细胞的50微升。接下来，将细胞培养物在4000倍G下离心5分钟。对于选择，丢弃上一提液，并将含有卡那霉素的五毫升 0.2 x LB 介质添加到携带选择标记的细胞中。在37摄氏度的摇床中孵育样品过夜。第二天，将隔夜培养盘上含有卡那霉素的0.2 x LB汞培养基，并在37摄氏度下孵育12小时。对于筛查，将装有筛查标记的合适数量的细胞板到含有适当抗生素的LB agar介质上。在37摄氏度下孵育8小时。在此之后，接种含有适当抗生素的LB介质与每个单组从板。在 37 摄氏度的摇床中孵育样品，同时在 250 rpm 时摇动。测量OD600和荧光，并计算荧光强度，如果使用GGFP RC。如果使用 PPG RC，测量细胞培养物的颜色发展。为了验证候选菌株的氨基酸成分，通过接种5毫升LB培养基来准备种子培养，每个候选菌株在250转/分和37摄氏度的摇床中过夜。第二天，在4000次G离心两分钟，从细胞培养的一毫升中收获细胞。丢弃上一杯，用一毫升无菌水重新暂停调色板。接种含有4%葡萄糖的M9介质20毫升，用200微升细胞悬浮液，在250毫升的摇床中孵育，转速为250 rpm，在37摄氏度下孵育24小时。第二天，将培养介质的一毫升在4000倍G下离心5分钟。将 200 微升的上清液转移到干净的 1.5 毫升管中。以此处所示的浓度准备 L-leucine 标准溶液。在烟罩中，在上流液和标准中加入100微升一毫摩尔三乙胺和100微升一摩尔苯甲氧乙烷。轻轻混合溶液，在室温下孵育一小时。然后，将400微升的n-hexane加入同一管，然后旋涡10秒。下相包含氨基酸衍生物，用于HPLC分析。通过0.2微升聚四氟乙烯膜过滤下相。在此之后，在配备 C18 柱的超 HPLC 上运行一微升样品，流速为每分钟 0.42 毫升，柱温为 40 摄氏度。使用二极管阵列检测器检测 254 纳米的靶向氨基酸，并绘制标准曲线下的峰值区域，计算其浓度。在这项研究中，使用罕见的富含共分的标记物来识别氨基酸超产剂，同时实现精度、灵敏度和高通量。对于选择系统，与野生型菌株相比，对含有稀有富含可发性抗生素耐药基因的菌株，应观察到OD600的急剧下降。对蛋白质表达的罕见蛋白的抑制大多发生在饥饿条件下。在额外喂养相应的氨基酸后，OD600为菌株，为含有罕见的富含可糖的抗生素耐药基因显著增加。在筛选系统中，荧光细胞数量中的荧光强度明显低于野生基因，即表达来自稀有富含科登基因的荧光蛋白的菌株。喂养相应的氨基酸将恢复蛋白质表达从罕见的富含科登的基因。使用紫色蛋白质时，从稀有富含共分的PPG中发展出的颜色比野生型基因的颜色要轻。未稀释的LB介质允许缓慢的表达罕见的富含共生丰富的PPG，没有L-leucine喂养在表达的紫色蛋白质变得可见，一旦细胞被造粒。然而，这并不掩盖这样一个事实，即通过喂养L-leucine，从罕见的富含糖的PPG的基因表达得到加强。罕见的基于科登的策略可以识别突变库中目标氨基酸的超产，这些突变体应产生比父菌株更多的靶向氨基酸。在尝试此程序时，必须调整稀有的 codon 频率，以实现在含有野生型标记的细胞和稀有的 codon 丰富的衍生物的细胞之间的 OD 或颜色上实现明显的区分。转录组分析和基因组测序可应用于选定的菌株，以研究与氨基酸生产相关的机制。利用这项技术，可以有效地识别氨基酸超产者，分析其遗传信息将提供新的见解，了解其背后的机制氨基酸过剩生产。氨基酸衍生可能是有害的。确保戴上手套和防护服，并在烟罩中执行这些步骤。

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

镍 - 席夫碱的水解复杂环境条件适宜酸不稳定保护基团的保留

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

从高梁叶组织中分离出希石，用于自上而下质量光谱分析潜在的表观遗传标记

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

使用硫二甘酸（TGA）估计植物生物质木质素含量

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...