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

Abstract

L-Fucose is a monosaccharide abundant in mammalian glycoconjugates. In humans, fucose can be found in human milk oligosaccharides (HMOs), mucins, and glycoproteins in the intestinal epithelium. The bacterial consumption of fucose and fucosylated HMOs is critical in the gut microbiome assembly of infants, dominated by Bifidobacterium. Fucose metabolism is important for the production of short-chain fatty acids and is involved in cross-feeding microbial interactions. Methods for assessing fucose concentrations in complex media are lacking. Here we designed and developed a molecular quantification method of free fucose using fluorescent Escherichia coli. For this, low- and high-copy plasmids were evaluated with and without the transcription factor fucR and its respective fucose-inducible promoter controlling the reporter gene sfGFP. E. coli BL21 transformed with a high copy plasmid containing pFuc and fucR displayed a high resolution across increasing fucose concentrations and high fluorescence/OD values after 18 h. The molecular circuit was specific against other monosaccharides and showed a linear response in the 0–45 mM range. Adjusting data to the Hill equation suggested non-cooperative, simple regulation of FucR to its promoter. Finally, the biosensor was tested on different concentrations of free fucose and the supernatant of Bifidobacterium bifidum JCM 1254 supplemented with 2-fucosyl lactose, indicating the applicability of the method in detecting free fucose. In conclusion, a bacterial biosensor of fucose was validated with good sensitivity and precision. A biological method for quantifying fucose could be useful for nutraceutical and microbiological applications, as well as molecular diagnostics.

Applications and Tuning Strategies for Transcription Factor-Based Metabolite Biosensors

“…The ability to sense and respond to intracellular metabolites is crucial for better understanding and regulating pathway behavior for enhanced metabolite production [ 1 ]. Many different types of biosensors have been developed and characterized in microbial hosts, including aptamers, riboswitches, fluorescence resonance energy transfer (FRET)-based sensors, and transcription factor (TF)-based sensors [ 2 , 3 , 4 , 5 ]. Aptamers are nucleic-acid-based biosensors that undergo a conformational change upon the binding of a target metabolite.…”