Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440

Abstract

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/PPP_RS00425 from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/PPP_RS00425 is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.

The development of an ingestible biosensor for the characterization of gut metabolites related to major depressive disorder: hypothesis and theory

“…Kinetic constants were sourced from the literature (Supplementary Table S1) (Bakshi et al, 2012;Bremer and Dennis, 2008;Hall et al, 2011;Krzysztoń et al, 2019;Liu et al, 2022;Matulis et al, 2022;Outten and O'Halloran, 2001;Raper et al, 2018;Tang and Liu, 2018;Yu et al, 2006).…”

“…We propose adopting the indole responsive gene expression system designed by Matulis et al (2022) for our ingestible biosensor as described in Figure 11.…”

“…Indole Sensing in Escherichia coli. Indole binds to PP_RS00430, inducing the expression of transcription factor (TrpI) allowing for the expression of tryptophan synthase subunits, now substituted with sgRNA located after the promoter PP_RS00425 (Matulis et al, 2022).…”

“…H2O2 Sensing in E. coli. Increased H2O2 levels result in the activation of the OxyR protein, resulting in the formation of a disulfide bond between two OxyR cysteine residues (Matulis et al, 2022). This activated OxyR state results in the activation of the TrxC promoter (pTrxC), leading to sgRNA expression.…”

Design and Characterization of a Generalist Biosensor for Indole Derivatives

“…Indole and indole derivatives are known for their biological and pharmacological activity, in addition to their roles in signaling and other processes as microbiome-produced metabolites. , There exists a small number of prokaryotic single-component TF-based biosensors targeting indole and indole derivatives. While the TF TrpI has been found to be highly specific for indole, it does not appear to be responsive to tested indole derivatives . The TF CmeR has been found to respond to indole acetic acid and tryptophol (indole-3-ethanol), although to a lesser extent than its ligand salicylate .…”