Journal Article

A Screening Assay for Bile Acid-Transforming Microorganisms Using Engineered Bacterial Biosensors

Dallera, Debora, Brun, Paola, Pasotti, Lorenzo, Usai, Francesca, Magni, Paolo, Cusella De Angelis, Maria Gabriella, Pastorelli, Daniele, Frusteri Chiacchiera, Angelica, Bellato, Massimo

2025

Overview

Bile salt hydrolase (BSH) enables microbial-mediated deconjugation of bile acids (BAs) in the gastrointestinal tract. BSH enzymes initiate bile acid metabolism by catalyzing the first, essential deconjugation step. Due to the strict connection between dysregulations of the BA pool and human or animal diseases, identification and characterization of strains with BSH activity are relevant for both healthcare and agroindustry. However, current methods are expensive, poorly sensitive, or require complex procedures. Here, a BSH screening assay for cultivated microbes is proposed, based on a bacterial biosensor that reports the concentration of different BA types via fluorescence. Although the biosensor is broadly responsive to various bile acids, the assay was designed to guarantee specificity by testing individual primary BAs within controlled concentration ranges. The assay was evaluated on two recombinant Escherichia coli strains bearing BSH genes from Lactobacillus johnsonii PF01 and a BSH-positive probiotic strain (Lactobacillus rhamnosus GG). Data showed a consistent activity pattern with previous assays on these enzymes. A crucial aspect addressed was the matrix effect, i.e., the impact of the growth media of the BSH-containing strains on biosensor output. This assay is expected to be a reproducible and accessible option, compatible with automated protocols.

• Publisher: MDPI AG
• Subject: Activity patterns / Agricultural industry / Amidohydrolases - metabolism / Amino acids / Animal diseases / Animal health / Assaying / bacterial sensor / Bile / Bile acids / Bile Acids and Salts - metabolism / bile salts / Biosensing Techniques - methods / Biosensors / Chromatography / Detectors / E coli / Enzymes / Escherichia coli / Escherichia coli - genetics / Escherichia coli - metabolism / Fluorescence / fluorimetric assay / Gastrointestinal system / Gastrointestinal tract / Genetic aspects / Glycerol / Growth media / Gut microbiota / Humans / Infections / lactic acid bacteria / Lactobacilli / Lactobacillus - enzymology / Lactobacillus - genetics / Medical equipment / Metabolism / Microorganisms / Physiological apparatus / Physiological aspects / Plasmids / Probiotics / specificity / Strains (organisms) / Toxicity