Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity.
Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity.
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DOI：
10.1021/cb3006402
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发表时间：
2013-04-19
影响因子：
4
通讯作者：
Collins, Cynthia H.
中科院分区：
文献类型：
--
作者：
Shong, Jasmine;Huang, Yao-Ming;Bystroff, Christopher;Collins, Cynthia H.
细胞间通讯或来自革兰氏阴性变形菌的“群体感应（QS）”元件的使用使得合成生物学家能够开始工程化由多种相互作用的生物体组成的系统。然而，如果我们要向表现出更复杂、动态行为的合成微生物财团迈进，额外的工具是必要的。EsaR来自Pantoea stewartii subsp. stewartii是一种QS调节剂，其作为脱辅基蛋白与DNA结合，并在与其同源信号分子3-氧代己酰基-高丝氨酸内酯（3 OC 6 HSL）结合时释放DNA。在缺乏3 OC 6 HSL的情况下，EsaR与DNA结合，可以作为转录的激活因子或阻遏因子。来自PesaR的基因表达被野生型EsaR抑制，需要比常用的QS激活剂（如LuxR和LasR）高100至1000倍的信号浓度。在这里，我们已经确定了EsaR的变异与增加的敏感性，30 C6 HSL使用定向进化和双ON/OFF筛选策略。尽管我们靶向了EsaR依赖的PesaR去阻遏，但我们的EsaR变体也显示出在第二启动子PesaS处增加的3 OC 6 HSL敏感性，PesaS在3 OC 6 HSL不存在的情况下被EsaR激活。在这里，阿勒敏感性的增加导致基因表达在较低浓度的3 OC 6 HSL下被关闭。总体而言，我们将EsaR的信号灵敏度提高了70倍以上，并生成了一组EsaR变体，可识别4个数量级以上的3 OC 6 HSL浓度。与DNA结合并在没有QS信号的情况下具有活性的QS依赖性转录调节因子代表了一组用于工程化细胞-细胞通信依赖性基因表达的新工具。
The use of cell–cell communication or “quorum sensing (QS)” elements from Gram-negative Proteobacteria has enabled synthetic biologists to begin engineering systems composed of multiple interacting organisms. However, additional tools are necessary if we are to progress toward synthetic microbial consortia that exhibit more complex, dynamic behaviors. EsaR from Pantoea stewartii subsp. stewartii is a QS regulator that binds to DNA as an apoprotein and releases the DNA when it binds to its cognate signal molecule, 3-oxohexanoyl-homoserine lactone (3OC6HSL). In the absence of 3OC6HSL, EsaR binds to DNA and can act as either an activator or a repressor of transcription. Gene expression from PesaR, which is repressed by wild-type EsaR, requires 100- to 1000-fold higher concentrations of signal than commonly used QS activators, such as LuxR and LasR. Here we have identified EsaR variants with increased sensitivity to 3OC6HSL using directed evolution and a dual ON/OFF screening strategy. Although we targeted EsaR-dependent derepression of PesaR, our EsaR variants also showed increased 3OC6HSL sensitivity at a second promoter, PesaS, which is activated by EsaR in the absence of 3OC6HSL. Here, the increase in AHL sensitivity led to gene expression being turned off at lower concentrations of 3OC6HSL. Overall, we have increased the signal sensitivity of EsaR more than 70-fold and generated a set of EsaR variants that recognize 3OC6HSL concentrations ranging over 4 orders of magnitude. QS-dependent transcriptional regulators that bind to DNA and are active in the absence of a QS signal represent a new set of tools for engineering cell–cell communication-dependent gene expression.
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影响因子：
9.9
作者：
Cox, Robert Sidney 3rd;Surette, Michael G;Elowitz, Michael B
通讯作者：
Elowitz, Michael B
影响因子：
2.8
作者：
Gray, KM;Garey, JR
通讯作者：
Garey, JR
影响因子：
64.5
作者：
ENGEBRECHT, J;NEALSON, K;SILVERMAN, M
通讯作者：
SILVERMAN, M
DOI：
10.1073/pnas.81.13.4154
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
ENGEBRECHT, J;SILVERMAN, M
通讯作者：
SILVERMAN, M
DOI：
10.1073/pnas.84.19.6639
发表时间：
1987-10-01
影响因子：
11.1
作者：
KAPLAN, HB;GREENBERG, EP
通讯作者：
GREENBERG, EP