基于转录因子PdhR的丙酮酸生物传感器的构建

Development of pyruvate biosensors based on the transcriptional factor PdhR

摘要:

丙酮酸是一种重要的有机酸，在食品、化工和医药等领域广泛应用。酶法合成丙酮酸存在酶活性低的问题，严重制约着丙酮酸的工业化生产。低通量的筛选方法是筛选高活性酶突变体的瓶颈，生物传感器能够为酶的分子改造提供高通量的筛选方法。本研究以大肠杆菌的转录因子PdhR为研究对象，构建响应丙酮酸的生物传感器。首先，将PdhR的结合位点插入3个启动子的不同位置构建杂合启动子并验证其响应活性，结果表明杂合启动子P_tacD、P_ticM和P_trpD具有较好的响应活性。其次，通过优化PdhR表达量改善生物传感器的响应性能，其中生物传感器B2-1能灵敏地响应丙酮酸的浓度变化，具有较宽的配体检测范围(0–12 g/L丙酮酸)，其荧光响应倍数为14。最后，生物传感器B2-1的配体特异性分析表明生物传感器B2-1仅响应丙酮酸，对其他酮酸不敏感，具有较强的配体特异性。因此，本研究构建的响应丙酮酸的生物传感器可用于高活性酶突变体的高通量筛选，为酶和菌种的分子改造提供了高效、灵敏的筛选工具，进一步为丙酮酸的工业化生产奠定了基础。

Abstract:

Pyruvate is an important organic acid applied in food, chemical, and pharmaceutical fields. The enzymatic synthesis of pyruvate faces the challenge of low enzyme activity, which seriously limits the industrial production of pyruvate. The low-throughput screening methods are the bottleneck for screening high-performance enzymes. Biosensors serve as a high-throughput screening method for molecular modification of enzymes. This study constructs biosensors responsive to pyruvate based on the pyruvate dehydrogenase complex regulator (PdhR) from Escherichia coli. Firstly, the binding site of PdhR was inserted into different sites of three promoters of E. coli to construct hybrid promoters, among which the hybrid promoters P_tacD, P_ticM, and P_trpD exhibited active responses to pyruvate. Secondly, the response performance of biosensors was improved by optimizing the expression level of PdhR. Among the constructed biosensors, B2-1 sensitively responded to changes in the concentration of pyruvate, with a wide ligand detection range (0–12 g/L pyruvate) and a luciferase induction fold change of 14. Finally, this study analyzed the ligand specificity of biosensor B2-1. The results showed that biosensor B2-1 only responded to pyruvate and was not sensitive to other ketone acids, indicating that this biosensor had strong ligand specificity. Therefore, the pyruvate biosensor constructed in this study can be used for high-throughput screening of highly active enzyme mutants, providing an efficient and sensitive screening tool for molecular modification of enzymes and strains, and further laying the foundation for the industrial production of pyruvate.