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

• Published in: Microbial cell factories Vol. 21; no. 1; pp. 66 - 15
• Main Authors: Kay, Emily J., Mauri, Marta, Willcocks, Sam J., Scott, Timothy A., Cuccui, Jon, Wren, Brendan W.
• Format: Journal Article
• Language: English
• Published: London BioMed Central 21.04.2022; BioMed Central Ltd; BMC
• Subjects: Antibiotics; Antigens; Applied Microbiology; Bacteria; Bacterial pneumonia; Bacterial Vaccines - genetics; Biological conjugation; Biopolymers; Biotechnology; Campylobacter; Capsular polysaccharides; Care and treatment; Chains; Chemistry; Chemistry and Materials Science; Chromosomes; Coliforms; Conjugates; Contaminants; CRISPR; Diagnosis; Dosage and administration; E coli; Endotoxins; Enzymology; Epimerase; Escherichia coli; Escherichia coli - metabolism; Genetic Engineering; Glucose; Glycan; Glycoconjugates; Glycoconjugates - metabolism; Glycoengineering; Lipids; Metabolic engineering; Metabolism; Methods; Microbial Genetics and Genomics; Microbial polysaccharides; Microbiology; Microorganisms; Mutation; PglB; Plasmids; Pneumonia; Polysaccharides; Polysaccharides - metabolism; Polysaccharides, Bacterial - metabolism; Properties; Proteins; Racemases and Epimerases - metabolism; Risk factors; Saccharides; Strains (organisms); Streptococcus infections; Streptococcus pneumoniae; Streptococcus pneumoniae - genetics; Streptococcus pneumoniae - metabolism; Testing; Vaccine; Vaccines; Vaccines, Conjugate; Denmark; Africa; PglB; Vaccine; Biological conjugation; Glycoconjugates; Glycoengineering; Streptococcus pneumoniae
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01792-7

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.