Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea.
Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Republic of Korea.
J Microbiol Biotechnol. 2023 Nov 28;33(11):1506-1512. doi: 10.4014/jmb.2305.05019. Epub 2023 Jul 27.
Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant , we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic using the CRISPR/Cas9-based genome editing system. The linear relationship (r = 0.9561) between ATP levels and bioluminescence generated from the engineered expressing FLuc was observed in vitro. To explore the feasibility of using the engineered expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (, ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.
三磷酸腺苷（ATP）的定量分析已广泛应用于食品和医疗行业的诊断工具。特别是，包括炎症性肠病（IBD）在内的一些疾病的发病机制与高浓度 ATP 密切相关。发光 D-荧光素/荧光素酶系统，其中包括萤火虫中的荧光素酶（FLuc）作为关键组成部分，是检测和定量 ATP 最常用的方法。在这里，我们的目标不是分离重组 中产生的 FLuc，而是开发一种不需要提取和纯化 FLuc 的全细胞生物催化剂系统。为此，使用基于 CRISPR/Cas9 的基因组编辑系统将编码 FLuc 的基因引入益生菌 的基因组中。在体外观察到表达 FLuc 的工程 产生的 ATP 水平与生物发光之间的线性关系（r = 0.9561）。为了探索表达 FLuc 的工程 作为全细胞生物传感器检测肠道中炎症生物标志物（ATP）的可行性，我们使用葡聚糖硫酸钠作为结肠炎化合物建立了结肠炎小鼠模型。我们的研究结果表明，全细胞生物传感器可以在小鼠肠道炎症期间检测到升高的 ATP 水平。因此，本文开发的简单而强大的方法可用于非侵入性 IBD 诊断。
