Chemical conjugation to carrier proteins has been traditionally used to improve polysaccharides immunogenicity and to overcome the limitations of T-independent antigens, including lack of immunological memory and no efficacy in infants. A double-hit approach, meaning that both polysaccharide and carrier protein belong to the same pathogen, may be particularly useful for bacterial species with large glycan variability. Recently, bacterial protein glycosylation has been exploited to obtain glycosylated proteins in E. coli cytoplasm. This system relies on a N-glucosyltransferase enzyme which catalyzes the transfer of a single β-linked glucose onto engineered N-x-S/T sequons on recombinant proteins. In my PhD work, I have used this technology for the development of novel selective glycoconjugates, with the aim of preserving the antigenicity of the carrier protein. Klebsiella pneumoniae (Kp) capsular (K-antigens) and subcapsular (O-antigens) polysaccharides have been selected as model antigens for the generation of double-hit vaccines using MrkA as carrier protein and potential Kp protective antigen. MrkA, the major component of Kp type 3 fimbriae, possesses a high degree of sequence conservation among different isolates, and thus its use in a glycoconjugate might increase the vaccine coverage, considering the diversity of Kp K- and O-antigens. A glycosylation pathway was successfully established in E. coli for MrkA modification with a lactose moiety, after deleting lacZ gene to prevent the disaccharide catabolism. Kp O1v1 O-antigen (~ 20 kDa) and K2 K-antigen (~ 150 kDa) were selectively conjugated to Lac-MrkA and their immunogenicity was evaluated in mice in comparison to more traditional random glycoconjugates as well as to MrkA alone. The ability of MrkA to work as carrier for polysaccharides was proportional to the molecular mass of the final glycoconjugate. Interestingly, the mice study highlighted the ability of a long polysaccharide like K2 to work as “carrier” for MrkA increasing the immunogenicity, poor per se, of the protein selectively linked along its chain. The new conjugation approach developed in this work can be easily extended to other pathogens, combining polysaccharide and protein antigens in novel effective glycoconjugate vaccines with broader coverage. Overall, my work represents an innovative example of how the glycoengineering technology can be combined to conjugation chemistry for the development of effective glycoconjugate vaccines.