Bhat Shrilaxmi, Banerjee Anantika, Alagesan Swathi
Institute of Bioinformatics and Applied Biotechnology (IBAB), Bengaluru 560100, India.
ACS Omega. 2023 Jul 18;8(30):26806-26815. doi: 10.1021/acsomega.3c01164. eCollection 2023 Aug 1.
Isoprene is a valuable platform chemical, which is produced by engineered microorganisms, albeit in low quantities. The amount of isoprene produced is usually measured by gas chromatography, which can be time-consuming and expensive. Alternatively, biosensors have evolved as a powerful tool for real-time high-throughput screening and monitoring of product synthesis. The AraC-pBAD-inducible system has been widely studied, evolved, and engineered to develop biosensors for small molecules. In our preliminary studies, the AraC-pBAD system was mildly induced at higher isoprene concentrations when arabinose was also available. Hence, in the present study, we designed and constructed a synthetic biosensor based on the AraC-pBAD system, wherein the ligand-binding domain of AraC was replaced with IsoA. On introducing this chimeric AraC-IsoA (AcIa) transcription factor with the native P promoter system regulating gene expression, fluorescence output was observed only when wild-type cells were induced with both isoprene and arabinose. The biosensor sensitivity and dynamic range were further enhanced by removing operator sequences and by substituting the native promoter (P) with the strong tac promoter (P). The chimeric sensor did not work in AraC knockout strains; however, functionality was restored by reintroducing AraC. Hence, AraC is essential for the functioning of our biosensor, while AcIa provides enhanced sensitivity and specificity for isoprene. However, insights into how AraC-AcIa interacts and the possible working mechanism remain to be explored. This study provides a prototype for developing chimeric AraC-based biosensors with proteins devoid of known dimerizing domains and opens a new avenue for further study and exploration.
异戊二烯是一种有价值的平台化学品，可由工程微生物生产，尽管产量较低。通常通过气相色谱法测量异戊二烯的产量，这可能既耗时又昂贵。另外，生物传感器已发展成为用于产品合成实时高通量筛选和监测的强大工具。AraC-pBAD诱导系统已得到广泛研究、改进和工程设计，以开发用于小分子的生物传感器。在我们的初步研究中，当也存在阿拉伯糖时，AraC-pBAD系统在较高异戊二烯浓度下被轻度诱导。因此，在本研究中，我们基于AraC-pBAD系统设计并构建了一种合成生物传感器，其中AraC的配体结合结构域被IsoA取代。在将这种嵌合的AraC-IsoA（AcIa）转录因子与调节基因表达的天然P启动子系统一起引入时，仅当野生型细胞用异戊二烯和阿拉伯糖同时诱导时才观察到荧光输出。通过去除操纵序列并用强tac启动子（P）替代天然启动子（P），进一步提高了生物传感器的灵敏度和动态范围。嵌合传感器在AraC基因敲除菌株中不起作用；然而，通过重新引入AraC恢复了功能。因此，AraC对于我们生物传感器的功能至关重要，而AcIa对异戊二烯具有更高的灵敏度和特异性。然而，关于AraC-AcIa如何相互作用以及可能的工作机制仍有待探索。本研究为开发基于嵌合AraC的生物传感器提供了一个原型，该生物传感器含有缺乏已知二聚化结构域的蛋白质，并为进一步的研究和探索开辟了一条新途径。
