Highly multiplexed design of an allosteric transcription factor to sense novel ligands

Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. Here we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

变构转录因子（Allosteric transcription factors, aTF）通过小分子结合诱导的构象变化调控基因表达。尽管这类蛋白已被广泛用作生物传感器，但设计用于检测新型分子的aTF仍颇具挑战——配体结合残基的突变往往会破坏变构效应。本研究开发了Sensor-seq技术，这是一种用于设计并识别可结合非天然配体的aTF生物传感器的高通量平台。我们针对6种结构各异的非天然配体（4种癌症治疗药物他莫昔芬衍生物、抗疟药奎宁以及阿片类类似物纳曲酮），以及2种天然类黄酮配体柚皮素和根皮素，对多药外排泵调控蛋白TtgR的17737个变体文库开展了筛选。Sensor-seq成功为每种配体鉴定出了具备高动态范围和多样特异性谱的新型生物传感器。纳曲酮结合型设计变体的结构显示，形状互补的甲硫氨酸-芳香族相互作用驱动了配体特异性。为验证实际应用价值，我们开发了针对纳曲酮和奎宁的无细胞检测系统。Sensor-seq可实现新型生物传感器的快速、规模化设计，突破了天然生物传感器的设计局限。