Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors
Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors
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DOI：
10.1021/acssynbio.0c00582
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发表时间：
2021-03-09
影响因子：
4.7
通讯作者：
Wilson, Corey J.
中科院分区：
文献类型：
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作者：
Rondon, Ronald;Wilson, Corey J.
合成生物学和蛋白质工程的最新进展增加了用于调节独立启动子的变构转录因子的数量。这些发展代表了我们生物计算能力的重要增长，这将使我们能够为广泛的生物技术构建更复杂的遗传程序。然而，这些转录因子中的大多数由阻遏物表型（BUFFER）表示，并且需要分层倒置以赋予对立逻辑功能（NOT），从而需要额外的生物资源。此外，这些工程化转录因子通常利用与替代DNA结合功能配对的天然配体结合功能。在这项研究中，我们通过工程化和重新设计PurR拓扑结构（一种天然抗阻遏物）来促进最先进的技术，以响应咖啡因，同时减轻对天然配体次黄嘌呤的响应-即，输入分子腺嘌呤的脱氨产物。重要的是，所产生的咖啡因响应转录因子不被天然配体次黄嘌呤拮抗。此外，我们赋予了与咖啡因抗阻遏物和PurR支架结合的替代DNA，创造了38个新的转录因子，这些转录因子与我们目前的转录编程结构一致。最后，我们利用这个转录因子系统来创建集成的NOR逻辑和相关的反馈操作。这项研究代表了第一个例子的转录因子（抗阻遏物）的系统中的配体结合位点和DNA结合功能被成功地串联工程。
Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to caffeine, while mitigating responsiveness to the native ligand hypoxanthine-i.e., a deamination product of the input molecule adenine. Importantly, the resulting caffeine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the caffeine antirepressors, and to the PurR scaffold, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the first example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.