Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
Abstract 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...
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| Format: | Article |
| Language: | English |
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BMC
2022-04-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-022-01792-7 |
| _version_ | 1818522693962563584 |
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| author | Emily J. Kay Marta Mauri Sam J. Willcocks Timothy A. Scott Jon Cuccui Brendan W. Wren |
| author_facet | Emily J. Kay Marta Mauri Sam J. Willcocks Timothy A. Scott Jon Cuccui Brendan W. Wren |
| author_sort | Emily J. Kay |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-11T05:36:29Z |
| format | Article |
| id | doaj.art-9f731b36816a4c29ae3d8c183184edb7 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-11T05:36:29Z |
| publishDate | 2022-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-9f731b36816a4c29ae3d8c183184edb72022-12-22T01:19:17ZengBMCMicrobial Cell Factories1475-28592022-04-0121111510.1186/s12934-022-01792-7Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccinesEmily J. Kay0Marta Mauri1Sam J. Willcocks2Timothy A. Scott3Jon Cuccui4Brendan W. Wren5Department of Infection Biology, London School of Hygiene & Tropical MedicineDepartment of Infection Biology, London School of Hygiene & Tropical MedicineDepartment of Infection Biology, London School of Hygiene & Tropical MedicineDepartment of Infection Biology, London School of Hygiene & Tropical MedicineDepartment of Infection Biology, London School of Hygiene & Tropical MedicineDepartment of Infection Biology, London School of Hygiene & Tropical MedicineAbstract 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.https://doi.org/10.1186/s12934-022-01792-7Biological conjugationStreptococcus pneumoniaeGlycoengineeringVaccinePglBGlycoconjugates |
| spellingShingle | Emily J. Kay Marta Mauri Sam J. Willcocks Timothy A. Scott Jon Cuccui Brendan W. Wren Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines Microbial Cell Factories Biological conjugation Streptococcus pneumoniae Glycoengineering Vaccine PglB Glycoconjugates |
| 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 | Biological conjugation Streptococcus pneumoniae Glycoengineering Vaccine PglB Glycoconjugates |
| url | https://doi.org/10.1186/s12934-022-01792-7 |
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