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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<jats:italic>E. coli<\/jats:italic>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In this study we have engineered a family of 11<jats:italic>E. coli<\/jats:italic>strains by the removal and\/or addition of components rationally selected for enhanced expression of<jats:italic>Streptococcus pneumoniae<\/jats:italic>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Amongst the 11 modified strains generated in this study,<jats:italic>E. coli<\/jats:italic>Falcon, Peregrine and Sparrowhawk all showed increased production of<jats:italic>S. pneumoniae<\/jats:italic>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<jats:italic>Streptococcus pneumoniae<\/jats:italic>) respectively produced an AcrA-SP4 conjugate with 4\u2009\u00d7\u2009and 14\u2009\u00d7\u2009more 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.<\/jats:p><\/jats:sec>","DOI":"10.1186\/s12934-022-01792-7","type":"journal-article","created":{"date-parts":[[2022,4,21]],"date-time":"2022-04-21T15:08:40Z","timestamp":1650553720000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines"],"prefix":"10.1186","volume":"21","author":[{"given":"Emily J.","family":"Kay","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Mauri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sam J.","family":"Willcocks","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy A.","family":"Scott","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jon","family":"Cuccui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6140-9489","authenticated-orcid":false,"given":"Brendan W.","family":"Wren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,4,21]]},"reference":[{"issue":"2","key":"1792_CR1","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1080\/02648725.2019.1703614","volume":"35","author":"TE MacCalman","year":"2019","unstructured":"MacCalman TE, Phillips-Jones MK, Harding SE. 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