Centre for Applied Synthetic Biology, Concordia University, Montréal, Québec, Canada.
Department of Biology, Concordia University, Montréal, Québec, Canada.
Nucleic Acids Res. 2023 Aug 11;51(14):7675-7690. doi: 10.1093/nar/gkad503.
Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.
重新编程细胞行为是合成生物学的标志之一。为此，原核变构转录因子（aTF）已被重新用作将小分子信号转化为细胞反应的多功能工具。扩展识别新诱导剂分子的 aTF 工具箱在许多应用中具有相当大的意义。在这里，我们首先使用来自谷氨酸棒杆菌的 TetR 家族抑制剂 RolR 在大肠杆菌中建立了基于间苯二酚的响应型 aTF 生物传感器。然后，我们沿着 RolR 的适应度景观进行迭代搜索，以确定新的诱导特异性，即儿茶酚、甲基儿茶酚、咖啡酸、原儿茶酸、L-DOPA 和肿瘤生物标志物高香草酸。最后，我们通过将这些工程化的 aTF 移植到模型真核生物酿酒酵母中来证明它们的多功能性。这项工作为高效的 aTF 工程提供了一个框架，可在实验室时间尺度上扩展配体特异性，以针对新型分子，更广泛地说，这在蛋白质和代谢工程以及即时诊断等广泛应用中具有不可估量的价值。
