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

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 inf...

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Autores principales: Nuñez, Samantha, Barra, Maria, Garrido, Daniel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046853/
https://www.ncbi.nlm.nih.gov/pubmed/36979599
http://dx.doi.org/10.3390/bios13030388
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author Nuñez, Samantha
Barra, Maria
Garrido, Daniel
author_facet Nuñez, Samantha
Barra, Maria
Garrido, Daniel
author_sort Nuñez, Samantha
collection PubMed
description 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.
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spelling pubmed-100468532023-03-29 Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli Nuñez, Samantha Barra, Maria Garrido, Daniel Biosensors (Basel) Article 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. MDPI 2023-03-15 /pmc/articles/PMC10046853/ /pubmed/36979599 http://dx.doi.org/10.3390/bios13030388 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Nuñez, Samantha
Barra, Maria
Garrido, Daniel
Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title_full Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title_fullStr Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title_full_unstemmed Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title_short Developing a Fluorescent Inducible System for Free Fucose Quantification in Escherichia coli
title_sort developing a fluorescent inducible system for free fucose quantification in escherichia coli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046853/
https://www.ncbi.nlm.nih.gov/pubmed/36979599
http://dx.doi.org/10.3390/bios13030388
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