Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology.

中文翻译：

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通过转座子介导的蛋白质融合开发新型代谢物反应转录因子。

自然进化的代谢物响应生物传感器可用于代谢工程，从筛选大型基因库到动态调节生物合成途径。然而，有许多代谢物不存在天然生物传感器。为了满足这一需求，我们开发了一种将代谢物结合蛋白转化为代谢物响应转录因子的通用方法——随机域插入生物传感器工程 (BERDI)。这种方法利用体外转座子插入反应生成所有可能的 DNA 结合结构域插入代谢物结合蛋白，然后进行荧光激活细胞分选以分离功能性生物传感器。为了开发和评估 BERDI 方法，我们生成了一个候选生物传感器库，其中将锌指 DNA 结合域插入到麦芽糖结合蛋白中，麦芽糖结合蛋白用作经过充分研究的代谢物结合蛋白模型。图书馆多样性的特点是有几种方法，部署了一个选择方案，并最终确定了几个不同的和功能性的麦芽糖响应转录生物传感器。我们假设 BERDI 方法包含一种可推广的策略，该策略最终可用于将各种代谢物结合蛋白转化为新型生物传感器，用于代谢工程和合成生物学。并最终确定了几种不同的和功能性的麦芽糖响应转录生物传感器。我们假设 BERDI 方法包含一种可推广的策略，该策略最终可用于将各种代谢物结合蛋白转化为新型生物传感器，用于代谢工程和合成生物学。并最终确定了几种不同的和功能性的麦芽糖响应转录生物传感器。我们假设 BERDI 方法包含一种可推广的策略，该策略最终可用于将各种代谢物结合蛋白转化为新型生物传感器，用于代谢工程和合成生物学。