Understanding and engineering solute transporters is important for metabolic engineering and the development of therapeutics. However, limited available experimental data on membrane transporters makes sequence-function relationships complex to predict. Here we apply ligand-responsive biosensor systems that enable selective growth of E. coli cells only if they functionally express an importer that is specific to the biosensor ligand. Using this system in a directed evolution framework, we successfully engineer the specificity of nicotinamide riboside transporters, PnuC, to accept thiamine as a substrate. Our results provide insight into the molecular determinants of substrate recognition of the PnuC transporter family and demonstrate how synthetic biology can be deployed to engineer the substrate spectrum of small molecule transporters.

中文翻译：

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使用合成选择的膜运输的定向进化

了解和工程化溶质转运蛋白对于代谢工程和治疗剂的开发很重要。然而，关于膜转运蛋白的有限的可用实验数据使得序列-功能关系难以预测。在这里，我们应用了能使大肠杆菌选择性生长的配体响应型生物传感器系统仅当细胞在功能上表达对生物传感器配体特异的导入物时才可以。在定向进化框架中使用该系统，我们成功地设计了烟酰胺核糖体转运蛋白PnuC的特异性，以接受硫胺素作为底物。我们的结果提供了对PnuC转运蛋白家族底物识别的分子决定因素的见解，并展示了如何利用合成生物学来设计小分子转运蛋白的底物谱。