Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase

  • Ji Sun Park (Department of Systems Biotechnology, Chung-Ang University) ;
  • Young-Woo Kim (Department of Food Science and Technology, Chung-Ang University) ;
  • Hyungdong Kim (Department of Food Science and Technology, Chung-Ang University) ;
  • Sun-Ki Kim (Department of Food Science and Technology, Chung-Ang University) ;
  • Kyeongsoon Park (Department of Systems Biotechnology, Chung-Ang University)
  • Received : 2023.05.19
  • Accepted : 2023.07.18
  • Published : 2023.11.28
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Abstract

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 Photinus pyralis 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 Escherichia coli, 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 Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., 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.

Keywords

Acknowledgement

This study was financially supported by the National Research Foundation of Korea Grant (2021R1A4A1022206), funded by the Korean Ministry of Science, ICT and Future Planning, and by a grant (21153MFDS605) from the Ministry of Food and Drug Safety in 2023. This research was also supported by the Chung-Ang University Graduate Research Scholarship (Academic Scholarship for the College of Biotechnology and Natural Resources) in 2023.

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