The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.

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基于配体依赖性稳定作用的大肠杆菌生物传感器策略

用于监测环境化学物质或产生所需生物产物的微生物工程通常依赖于合适的生物传感器的可用性。然而，将配体结合蛋白转化为生物传感器是困难的。在这里，我们报告了一种在大肠杆菌中产生生物传感器的一般策略，该传感器通过转录激活剂的配体依赖性稳定作用和介导报告基因的配体浓度依赖性表达而起作用。我们通过使用lac阻遏物LacI作为配体结合结构域并将其融合到Zif268 DNA结合结构域和RNA聚合酶ω亚基转录激活结构域来构建这样的生物传感器。使用容易出错的lacI PCR诱变和选择，我们确定了具有多个突变的生物传感器，其中只有一个对于生物传感器的行为是必不可少的。通过调整测定的参数，我们获得了取决于配体异丙基β- d -1-硫代半乳糖吡喃糖苷（IPTG）的响应，该响应的大肠杆菌生长速率最多可提高7倍。单个破坏稳定性的突变与lacI突变（将配体特异性扩展至d-岩藻糖）相结合，产生了对d响应均得到改善的生物传感器-fucose和IPTG。但是，与在两个结构相似的周质结合蛋白中的任何一个使LacI不稳定的突变相同的突变不会赋予配体依赖性稳定作用。最后，我们通过诱变和选择来工程化另一个配体结合域MphR以充当生物传感器，证明了该方法的一般性。这种策略可以使许多识别并结合配体的天然蛋白质转化为生物传感器。