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Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines

BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacteri...

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Autores principales: Kay, Emily J., Mauri, Marta, Willcocks, Sam J., Scott, Timothy A., Cuccui, Jon, Wren, Brendan W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026721/
https://www.ncbi.nlm.nih.gov/pubmed/35449016
http://dx.doi.org/10.1186/s12934-022-01792-7
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author Kay, Emily J.
Mauri, Marta
Willcocks, Sam J.
Scott, Timothy A.
Cuccui, Jon
Wren, Brendan W.
author_facet Kay, Emily J.
Mauri, Marta
Willcocks, Sam J.
Scott, Timothy A.
Cuccui, Jon
Wren, Brendan W.
author_sort Kay, Emily J.
collection PubMed
description BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse. RESULTS: In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences. CONCLUSIONS: Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01792-7.
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spelling pubmed-90267212022-04-23 Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines Kay, Emily J. Mauri, Marta Willcocks, Sam J. Scott, Timothy A. Cuccui, Jon Wren, Brendan W. Microb Cell Fact Research BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse. RESULTS: In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences. CONCLUSIONS: Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01792-7. BioMed Central 2022-04-21 /pmc/articles/PMC9026721/ /pubmed/35449016 http://dx.doi.org/10.1186/s12934-022-01792-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Kay, Emily J.
Mauri, Marta
Willcocks, Sam J.
Scott, Timothy A.
Cuccui, Jon
Wren, Brendan W.
Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title_full Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title_fullStr Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title_full_unstemmed Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title_short Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
title_sort engineering a suite of e. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026721/
https://www.ncbi.nlm.nih.gov/pubmed/35449016
http://dx.doi.org/10.1186/s12934-022-01792-7
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