Screening of an Escherichia coli promoter library for a phenylalanine biosensor
Screening of an Escherichia coli promoter library for a phenylalanine biosensor
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DOI：
10.1007/s00253-016-7575-8
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发表时间：
2016-08-01
影响因子：
5
通讯作者：
Frunzke, Julia
中科院分区：
文献类型：
--
作者：
Mahr, Regina;von Boeselager, Raphael Freiherr;Frunzke, Julia
近年来，基于转录因子的生物传感器在微生物生产菌株工程中的应用为工业生物技术开辟了新的机遇。然而，合成调节电路的设计依赖于选择合适的转录因子-启动子对来将效应分子的浓度转化为可测量的输出。在这里，我们提出了一种有效的策略来筛选启动子文库，为生物传感器的设计提供合适的部分。为此，我们汇集了包含约2000个不同的大肠杆菌启动子-gfpmu2融合基因的ALON文库中的菌株，并通过荧光激活细胞分类的正反两轮选择，丰富了半乳糖和L-苯丙氨酸反应启动子。对于这两种效应子，都分离出了响应性启动子，并通过微量平板培养进行了验证。编码L-色氨酸特异性转运蛋白的mtr启动子被确定为构建L-苯丙氨酸生物传感器的合适部分。在下文中，我们对基于MTR启动子的不同生物传感器构建进行了比较分析。实验数据表明，生物传感器的结构对其性能有很大的影响。为了验证原理，将MTR传感器应用于流式细胞仪高通量筛选E.ColiMG1655突变体库，以分离L-苯丙氨酸产生菌。这些结果强调了所开发的筛选方法作为一种方便的策略来识别用于设计新型生物传感器的效应型启动子。
In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose-and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.