Georgia Institute of Technology, School of Chemical & Biomolecular Engineering, 311 Ferst Drive, Atlanta, Georgia 30332-0100, United States.
ACS Synth Biol. 2021 Mar 19;10(3):552-565. doi: 10.1021/acssynbio.0c00582. Epub 2021 Mar 9.
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-, 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.
近年来，合成生物学和蛋白质工程的进展增加了用于调节独立启动子的别构转录因子的数量。这些发展代表了我们生物计算能力的重要提高，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都代表了抑制表型（BUFFER），并且需要分层反转来赋予对偶逻辑功能（NOT），这需要额外的生物资源。此外，这些工程化的转录因子通常利用天然配体结合功能与替代 DNA 结合功能配对。在这项研究中，我们通过对 PurR 拓扑结构（天然反阻遏物）进行工程改造和重新设计，使其对咖啡因产生反应，同时减轻对天然配体次黄嘌呤（输入分子腺嘌呤的脱氨产物）的反应性，从而将这一技术提升到了新的水平。重要的是，由此产生的对咖啡因有反应的转录因子不受天然配体次黄嘌呤的拮抗。此外，我们赋予了咖啡因反阻遏物和 PurR 支架替代的 DNA 结合功能，从而创建了 38 个新的转录因子，与我们当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 NOR 逻辑和相关反馈操作。这项研究代表了第一个系统的转录因子（反阻遏物）的例子，其中配体结合位点和 DNA 结合功能都成功地串联进行了工程改造。
