Transcription factor (TF)-based biosensors have proven useful for increasing biomanufacturing yields, large-scale functional screening, and in environmental monitoring. Most yeast TF-based biosensors are built from natural promoters, resulting in large DNA parts retaining considerable homology to the host genome, which can complicate biological engineering efforts. There is a need to explore smaller, synthetic biosensors to expand the options for regulating gene expression in yeast. Here, we present a systematic approach to improving the design of an existing oxidative stress sensing biosensor in Saccharomyces cerevisiae based on the Yap1 transcription factor. Starting from a synthetic core promoter, we optimized the activity of a Yap1-dependent promoter through rational modification of a minimalist Yap1 upstream activating sequence. Our novel promoter achieves dynamic ranges of activation surpassing those of the previously engineered Yap1-dependent promoter, while reducing it to only 171 base pairs. We demonstrate that coupling the promoter to a positive-feedback-regulated TF further improves the biosensor by increasing its dynamic range of activation and reducing its limit of detection. We have illustrated the robustness and transferability of the biosensor by reproducing its activity in an unconventional probiotic yeast strain, Saccharomyces boulardii. Our findings can provide guidance in the general process of TF-based biosensor design.

中文翻译：

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改进酵母氧化应激传感生物传感器的设计

基于转录因子 (TF) 的生物传感器已被证明可用于提高生物制造产量、大规模功能筛选和环境监测。大多数基于酵母 TF 的生物传感器是由天然启动子构建的，导致大的 DNA 部分与宿主基因组保持相当大的同源性，这会使生物工程工作复杂化。有必要探索更小的合成生物传感器，以扩大在酵母中调节基因表达的选择。在这里，我们提出了一种系统方法来改进基于 Yap1 转录因子的酿酒酵母中现有的氧化应激传感生物传感器的设计。从合成核心启动子开始，我们通过合理修改极简 Yap1 上游激活序列来优化 Yap1 依赖性启动子的活性。我们的新型启动子实现了超过先前设计的 Yap1 依赖性启动子的动态激活范围，同时将其减少到仅 171 个碱基对。我们证明了将启动子与正反馈调节的 TF 偶联通过增加其激活的动态范围和降低其检测限来进一步改进生物传感器。我们通过在非常规益生菌酵母菌株布拉氏酵母中复制其活性来说明生物传感器的稳健性和可转移性。我们的研究结果可以为基于 TF 的生物传感器设计的一般过程提供指导。我们证明了将启动子与正反馈调节的 TF 偶联通过增加其激活的动态范围和降低其检测限来进一步改进生物传感器。我们通过在非常规益生菌酵母菌株布拉氏酵母中复制其活性来说明生物传感器的稳健性和可转移性。我们的研究结果可以为基于 TF 的生物传感器设计的一般过程提供指导。我们证明了将启动子与正反馈调节的 TF 偶联通过增加其激活的动态范围和降低其检测限来进一步改进生物传感器。我们通过在非常规益生菌酵母菌株布拉氏酵母中复制其活性来说明生物传感器的稳健性和可转移性。我们的研究结果可以为基于 TF 的生物传感器设计的一般过程提供指导。