Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.
ACS Synth Biol. 2021 Aug 20;10(8):2002-2014. doi: 10.1021/acssynbio.1c00165. Epub 2021 Aug 9.
The time-consuming and laborious characterization of protein or microbial strain designs limits the development of high-performance biocatalysts for biotechnological applications. Here, transcriptional biosensors emerged as valuable tools as they allow for rapid characterization of several thousand variants within a very short time. However, for many molecules of interest, no specific transcriptional regulator determining a biosensor's specificity is available. We present an approach for rapidly engineering biosensor specificities using a semirational transition ligand approach combined with fluorescence-activated cell sorting. In this two-step approach, a biosensor is first evolved toward a more relaxed-ligand specificity before using the resulting variant as the starting point in a second round of directed evolution toward high specificity for several chemically different ligands. By following this strategy, highly specific biosensors for 4-hydroxybenzoic acid, -coumaric acid, 5-bromoferulic acid, and 6-methyl salicylic acid were developed, starting from a biosensor for the intracellular detection of -cinnamic acid.
蛋白质或微生物菌株设计的耗时和费力的特征描述限制了用于生物技术应用的高性能生物催化剂的发展。在这里，转录生物传感器作为有价值的工具出现，因为它们允许在非常短的时间内快速表征数千个变体。然而，对于许多感兴趣的分子，没有可用的特定转录调节剂来确定生物传感器的特异性。我们提出了一种使用半理性过渡配体方法结合荧光激活细胞分选来快速工程生物传感器特异性的方法。在这个两步法中，首先使生物传感器朝着更宽松的配体特异性进化，然后使用所得变体作为第二轮针对几种化学上不同的配体的高特异性定向进化的起点。通过遵循这种策略，从用于检测细胞内 -肉桂酸的生物传感器开始，开发出了对 4-羟基苯甲酸、-香豆酸、5-溴阿魏酸和 6-甲基水杨酸具有高度特异性的生物传感器。
