Abstract
AbstractBackgroundGlycoengineering, 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 suitableE. colihost 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.ResultsIn this study we have engineered a family of 11E. colistrains by the removal and/or addition of components rationally selected for enhanced expression ofStreptococcus pneumoniaecapsular 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.ConclusionsAmongst the 11 modified strains generated in this study,E. coliFalcon, Peregrine and Sparrowhawk all showed increased production ofS. pneumoniaeserotype 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 fromStreptococcus 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.
Funder
Biotechnology and Biological Sciences Research Council
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献