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Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose

BACKGROUND: The trisaccharide 2(′)-fucosyllactose (2(′)-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2(′)-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk...

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Autores principales: Baumgärtner, Florian, Seitz, Lyudmila, Sprenger, Georg A, Albermann, Christoph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655002/
https://www.ncbi.nlm.nih.gov/pubmed/23635327
http://dx.doi.org/10.1186/1475-2859-12-40
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author Baumgärtner, Florian
Seitz, Lyudmila
Sprenger, Georg A
Albermann, Christoph
author_facet Baumgärtner, Florian
Seitz, Lyudmila
Sprenger, Georg A
Albermann, Christoph
author_sort Baumgärtner, Florian
collection PubMed
description BACKGROUND: The trisaccharide 2(′)-fucosyllactose (2(′)-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2(′)-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2(′)-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2(′)-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2(′)-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2(′)-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. RESULTS: Here we report the construction of the first selection marker-free E. coli strain that produces 2(′)-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2(′)-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2(′)-FL. Using an improved production strain, feasibility of large scale 2(′)-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2(′)-FL concentration of 20.28 ± 0.83 g l(-1) and a space-time-yield of 0.57 g l(-1) h(-1). CONCLUSIONS: By chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2(′)-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2(′)-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2(′)-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2(′)-FL could be demonstrated.
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spelling pubmed-36550022013-05-20 Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose Baumgärtner, Florian Seitz, Lyudmila Sprenger, Georg A Albermann, Christoph Microb Cell Fact Research BACKGROUND: The trisaccharide 2(′)-fucosyllactose (2(′)-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2(′)-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2(′)-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2(′)-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2(′)-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2(′)-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. RESULTS: Here we report the construction of the first selection marker-free E. coli strain that produces 2(′)-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2(′)-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2(′)-FL. Using an improved production strain, feasibility of large scale 2(′)-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2(′)-FL concentration of 20.28 ± 0.83 g l(-1) and a space-time-yield of 0.57 g l(-1) h(-1). CONCLUSIONS: By chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2(′)-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2(′)-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2(′)-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2(′)-FL could be demonstrated. BioMed Central 2013-05-01 /pmc/articles/PMC3655002/ /pubmed/23635327 http://dx.doi.org/10.1186/1475-2859-12-40 Text en Copyright © 2013 Baumgärtner et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Baumgärtner, Florian
Seitz, Lyudmila
Sprenger, Georg A
Albermann, Christoph
Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title_full Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title_fullStr Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title_full_unstemmed Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title_short Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
title_sort construction of escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2(′)-fucosyllactose
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655002/
https://www.ncbi.nlm.nih.gov/pubmed/23635327
http://dx.doi.org/10.1186/1475-2859-12-40
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