Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• L-fucose is a crucial monosaccharide in mammalian glycoconjugates, including human milk oligosaccharides (HMOs) and gut glycoproteins.
• Bacterial metabolism of fucose significantly influences infant gut microbiome development and microbial cross-feeding interactions.
• Current methods for quantifying fucose in complex biological or microbiological media are limited.

Purpose of the Study:

• To design and validate a novel molecular method for quantifying free L-fucose concentrations.
• To develop a sensitive and specific bacterial biosensor for fucose detection.

Main Methods:

• Engineered fluorescent *Escherichia coli* strains utilizing plasmids with fucose-inducible promoters (pFuc) and the transcription factor fucR.
• Evaluated high- and low-copy plasmids, optimizing for reporter gene (sfGFP) expression and fluorescence/optical density (OD) ratios.
• Tested biosensor specificity against other monosaccharides and its response linearity across a range of fucose concentrations (0-45 mM).

Main Results:

• A high-copy plasmid system with pFuc and fucR in *E. coli* BL21 demonstrated high resolution and sensitivity for fucose quantification after 18 hours.
• The developed biosensor exhibited specificity for fucose over other monosaccharides and a linear response within the tested concentration range.
• The biosensor successfully detected free fucose in pure solutions and in the supernatant of *Bifidobacterium bifidum* cultured with fucosylated substrates.

Conclusions:

• A sensitive and precise bacterial biosensor for L-fucose quantification has been successfully developed and validated.
• This biological method holds potential for applications in nutraceuticals, microbiology, and molecular diagnostics.
• The biosensor's ability to function in complex samples like bacterial supernatants highlights its practical utility.