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Directed evolution of VanR biosensor specificity in yeast 酵母中VanR生物传感器特异性的定向进化

Biotechnology Notes

Pub Date : 2020-01-01 DOI: 10.1016/j.biotno.2020.01.002

Vasil D'Ambrosio , Subrata Pramanik , Kati Goroncy , Tadas Jakočiūnas , David Schönauer , Mehdi D. Davari , Ulrich Schwaneberg , Jay D. Keasling , Michael K. Jensen

Allosterically regulated transcription factors (aTFs) based biosensors from prokaryotes have been widely used to screen large gene libraries, stabilize engineered microbes from evolutionary drifting, and for detection of soil pollutants, among many other applications. However, even though aTF-based biosensors have been established as successful tools for bioengineering and remediation, rational engineering of aTF small molecule-specificity have so far not been demonstrated, highlighting the need for a deeper understanding of the sequence-function relationships that govern aTF allostery. Here, by combining directed evolution of a naïve library of VanR, a vanillic acid transcriptional regulator from Caulobacter crescentus in yeast, followed by saturation mutagenesis of selected positions we identify residues required for vanillic acid responsiveness, while at the same time maintaining responsiveness to vanillin. Selected single-position VanR mutants show both complete repression of transcription in the absence of any ligand, complete loss of vanillic acid responsiveness, while still maintaining high derepression in the presence of vanillin. By computational ligand docking analyses we also discuss the structure-function relationship single mutations can have on aTF specificity, an attribute potentially accounting for the wide-spread arise of aTF members belonging to the GntR superfamily of transcriptional regulators.

基于变结构调节转录因子(aTFs)的原核生物传感器已广泛用于筛选大型基因文库，稳定进化漂移的工程微生物，以及土壤污染物的检测，以及许多其他应用。然而，尽管基于aTF的生物传感器已被建立为生物工程和修复的成功工具，但aTF小分子特异性的合理工程迄今尚未得到证实，这突出表明需要更深入地了解控制aTF变质的序列-功能关系。在这里，通过结合VanR(一种来自酵母中crescent Caulobacter的香兰酸转录调节因子)naïve文库的定向进化，然后对选定的位置进行饱和诱变，我们确定了香兰酸响应所需的残基，同时保持对香兰素的响应。选择的单位置VanR突变体在没有任何配体的情况下表现出完全的转录抑制，完全丧失对香草酸的反应，同时在香兰素存在时仍然保持高度的抑制。通过计算配体对接分析，我们还讨论了单个突变对aTF特异性的结构-功能关系，这一属性可能解释了属于转录调节因子GntR超家族的aTF成员广泛出现的原因。

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引用次数: 12

Copper-tripeptide complexes for rapid inactivation of Bacillus subtilis endospores 铜-三肽复合物快速灭活枯草芽孢杆菌孢子内

Biotechnology Notes

Pub Date : 2020-01-01 DOI: 10.1016/j.biotno.2020.03.002

Yonghao Cui, Kang Zhou, Kun-Lin Yang

Bacterial endospores can be pathogenic to human beings. However, they are robust and thus difficult to kill due to their rigid structure. Conventional methods such as autoclaving for inactivating endospores are energy-intensive and time-consuming. In this study, we developed a copper-tripeptide complexes reagent composed of copper-tripeptide complexes, hydrogen peroxide, and cetyl trimethylammonium bromide (CTAB), to kill endospores. This copper-tripeptide complexes reagent can achieve a 10^7.8-fold reduction in viable endospore count after 60-min treatment at ambient conditions. As a cost-effective and stable sporicidal agent, this reagent may be applied as a general-purpose disinfectant and a replacement of standard sterilization procedures in the future.

细菌内生孢子对人类具有致病性。然而，由于它们的刚性结构，它们很强壮，因此很难杀死。传统的方法，如高压灭菌灭活内生孢子是能源密集型和耗时的。在这项研究中，我们开发了一种铜-三肽配合物试剂，由铜-三肽配合物、过氧化氢和十六烷基三甲基溴化铵(CTAB)组成，以杀死内生孢子。这种铜-三肽复合物试剂在环境条件下处理60分钟后，可使活孢子数减少107.8倍。作为一种具有成本效益和稳定性的杀孢剂，该试剂可作为通用消毒剂应用，并在未来替代标准灭菌程序。

引用次数: 1

Improving Spinach2-and Broccoli-based biosensors for single and double analytes 改进基于菠菜2和西兰花的单、双分析生物传感器

Biotechnology Notes

Pub Date : 2020-01-01 DOI: 10.1016/j.biotno.2020.01.001

Shuo-Fu Yuan , Hal S. Alper

The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used in vivo for cell sensing and in vitro for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNA^Lys3 or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding in vitro and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.

近年来，利用基于“菠菜”的RNA传感器检测小代谢物和蛋白质受到越来越多的关注。虽然这种方法可以在体内用于细胞传感和在体外用于微流控分析，但由于对镁离子的高度依赖，它们的整体效用受到限制。在这里，我们通过结合人类tRNALys3或工程病毒F30(一种三向连接RNA基序)支架来缓解这一限制，以促进体外适体折叠，并提高所选链霉亲和素、酪氨酸和凝血酶适体的性能。此外，我们演示了将西兰花适体支架与病毒F30结合使用，以逻辑门类型的方式同时检测两个分子。我们的概念验证结果证明了重新设计这些适体传感器的能力，以提高亮度和信号稳定性，而不需要高镁，这两种特性都可以进一步增强下游筛选应用。

引用次数: 4

Directed evolution of VanR biosensor specificity in yeast 酵母中VanR生物传感器特异性的定向进化

Biotechnology Notes

Pub Date : 2020-01-01 DOI: 10.1016/j.biotno.2020.01.002

Vasil D'ambrosio, S. Pramanik, Kati Goroncy, Tadas Jakočiūnas, David Schönauer, M. Davari, U. Schwaneberg, J. Keasling, M. K. Jensen

引用次数: 15

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