Development of a novel glycoengineering platform for the rapid production of conjugate vaccines

Abstract

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

A platform for the recombinant production of Group A Streptococcus glycoconjugate vaccines

“…We next aimed to determine whether Shi1/Shi2a engineered RhaPS would be recognised as substrates by Cj PglB. Plasmids encoding the RhaPS biosynthesis genes and the Shi1 or Shi2 repeating unit pathways were transformed into an E. coli strain that harboured a chromosomal, IPTG-inducible copy of cjpglB [53, 54].…”

“…Transformed cells were grown at 37°C overnight in LB media, induced with 0.1 mM IPTG and 0.2% arabinose. E. coli MAGIC cells containing a chromosomal copy of the pglB gene under an IPTG inducible promotor ( cedA :: pglB ) [53, 54] were made electro- or chemically competent in LB media containing Kanamycin (50 µg/mL) and transformed with 10-100 ng of plasmids of interest (Table 1) and selected on LB agar supplemented with the corresponding selection antibiotics Kanamycin (K, 50 µg/mL), Ampicillin (A, 100 µg/mL), Spectinomycin (S, 80 µg/mL) and Erythromycin (E, 150 µg/mL), KASE. One to five colonies were swabbed of the plate and grown overnight in LB media with the required antibiotics at 37°C.…”

A combinatorial DNA assembly approach to biosynthesis ofN-linked glycans inE. coli

“…1 and 2A ) was deliberately excluded as it would offer more flexibility as to which transferase could be used for coupling to an acceptor protein. Furthermore, the notion that the expression of pglB could be detrimental to the host cell has been previously reported ( Abouelhadid et al 2021 ), and we treated it as a separate engineering challenge for the purpose of this study. Each CDS was searched for BsaI, SapI, and BbsI restriction sites, and those genes which did not contain these restriction sites were amplified by PCR from C. jejuni genomic DNA.…”

“…To determine whether these refactored loci would also improve the yield of glycoconjugate, the 8 clones previously selected were transformed into an E. coli strain that was specifically engineered as a glycoconjugate production platform. This chassis strain is derived from the waaL -, wecA + strain, CLM24, and expresses a chromosomal copy of the C. jejuni OST pglB ( Abouelhadid et al 2021 ) . The carrier protein ExoA from Pseudomonas aeruginosa was encoded on a second plasmid and harbors two PglB glycosylation sequons.…”