基于转录调节因子PuuR的1,4-丁二胺生物传感器的构建与优化

Construction and optimization of 1,4-butanediamine biosensor based on transcriptional regulator PuuR

摘要:

生物传感器已经成为实时监测特定小分子和精确控制生物系统中基因表达的强大工具。用于1,4-丁二胺生物合成的高通量传感器可以极大地提高1,4-丁二胺高产菌株的筛选效率。为研究调整生物传感器特性的策略，本研究开发了一种以转录调节因子PuuR为基础的1,4-丁二胺生物传感器，其同源的操作子puuO被组装在大肠杆菌组成型启动子P_gapA中，以控制下游的高能绿色荧光蛋白(superfolder green fluorescent protein,sfGFP)作为报告蛋白表达。最终该传感器在1,4-丁二胺浓度处于0–50 mmol/L时GFP/OD₆₀₀值与1,4-丁二胺浓度之间能稳定地表现出线性关系。本研究采用大肠杆菌基因组中不同强度的启动子对1,4-丁二胺生物传感器进行分子改造，探究并改进基于PuuR的1,4-丁二胺生物传感器的功能性质，为高通量筛选高产1,4-丁二胺的工程菌株奠定了基础。

关键词:大肠杆菌;1,4-丁二胺;生物传感器;转录调节因子PuuR

Abstract:

Biosensors have become powerful tools for real-time monitoring of specific small molecules and precise control of gene expression in biological systems. High-throughput sensors for 1,4-butanediamine biosynthesis can greatly improve the screening efficiency of high-yielding 1,4-butanediamine strains. However, the strategies for adapting the characteristics of biosensors are still rarely studied, which limits the applicability of 1,4-butanediamine biosensors. In this paper, we propose the development of a 1,4-butanediamine biosensor based on the transcriptional regulator PuuR, whose homologous operator puuO is installed in the constitutive promoter P_gapA of Escherichia coli to control the expression of the downstream superfolder green fluorescent protein (sfGFP) as the reporter protein. Finally, the biosensor showed a stable linear relationship between the GFP/OD₆₀₀ value and the concentration of 1,4-butanediamine when the concentration of 1,4-butanediamine was 0–50 mmol/L. The promoters with different strengths in the E.coligenome were used to modify the 1,4-butanediamine biosensor, and the functional properties of the PuuR-based 1,4-butanediamine biosensor were explored and improved, which laid the groundwork for high-throughput screening of engineered strains highly producing 1,4-butanediamine.

Key words:Escherichia coli;1,4-butanediamine;biosensor;transcriptional regulator PuuR