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First 5000 Characters: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.
The alarming rise in antimicrobial resistance necessitates global efforts to prevent a future health crisis. For more than half a century, antibiotics were considered the first line of defence against bacterial pathogens (1) .
However, the spread of antibiotic resistance amongst pathogenic bacteria entails considerable efforts to look for antibiotic alternatives. Vaccines have been successful in curbing infectious diseases for decades, not only among adults but also among children and the elderly, thus saving millions of lives worldwide (2) . According to the Market Information for Access to Vaccines (MI4A), World Health Organization, the vaccines market is estimated to be worth approximately $33 billion in 2019 (3). Current biotechnological platforms however, might not be able to fulfil the vaccines supply demand.
To satisfy the market's demand for conjugate vaccines, to protect humanity from a foreseeable pandemic, and to be able to tailor novel efficacious vaccines at lower cost, significant biotechnological innovation is needed.
Glycoconjugate vaccines are considered to be one of the safest and most effective tools to combat serious infectious diseases including bacterial meningitis and pneumonia (4) . Conjugation is achieved by linking glycans (carbohydrate moiety), either chemically or enzymatically, to proteins via covalent bonds. This leads to a T-cell dependent immune response, offering excellent protection in people of all ages (5) . Traditionally chemical approaches to produce glycoconjugate vaccine involve the activation of functional groups on the glycan and protein that are linked chemically in a multi-step method that is expensive and laborious, requiring several rounds of purification after each step (6) . Additionally, chemical conjugation methods such as reductive amination can alter the polysaccharide epitope, affecting the immunogenicity of the glycoconjugate against the disease, besides its inherent batch-to-batch variation (7) . Biological conjugation (bioconjugation) offers an excellent alternative to chemical conjugation. It is based on using a bacterial cell, usually E. coli, as a chassis to express a pathway that encodes the desired bacterial polysaccharide, carrier protein, and an oligosaccharytransferase enzyme, OST, that catalyses the conjugation process (6) .
The advent of the bacterial bioconjugation method allowed several protein glycan vaccine combinations to be successfully developed, emphasizing its immense potential to become the preferred method to develop glycoconjugate vaccines in the future (4, (8) (9) (10) (11) (12) . However, several challenges remain. Firstly, the process places significant metabolic stress on the E. coli cell, vaccine micro-factory, due to the expression of orthogonal pathways (13) . This process requires the prior genetic and structural information of the polysaccharide structure of choice. Secondly, the use of three independent replicons has limitations due to incompatibility of plasmid origins of replication and antibiotic selection markers which may lead to the plasmid loss that results in reduction in glycoconjugate yield (8, 10, 14) . Thirdly, reports have demonstrated that the expression of the OST PglB, that catalyses the linking of glycans to carrier proteins, has a detrimental effect on bacterial growth, thus decreasing cellular fitness to produce glycoconjugates (13, 15) . All this together results in a low biomass which often translates to a reduction in the vaccine yield. Consequently, this leads to an increase in the production cost of a glycoconjugate vaccine, making it unaffordable in low-income countries where they are most needed, putting millions of lives at risk as a result of vaccines inequity (6) .
Previous attempts to engineer robust glycoengineering host stra