Vaccination is considered the most effective means to fight against the multidrug-resistant strains ofKlebsiella pneumoniae. In recent years, a potential protein glycan coupling technology has been extensively used in the production of bioconjugated vaccines. Here, a series of glycoengineering strains derived fromK. pneumoniaeATCC 25955 were designed for protein glycan coupling technology. The capsule polysaccharide biosynthesis gene cluster and the O-antigen ligase genewaaLwere deleted via the CRISPR/Cas9 system to further weaken the virulence of host stains and block the unwanted endogenous glycan synthesis. Particularly, the SpyCatcher protein in the efficient protein covalent ligation system (SpyTag/SpyCatcher) was selected as the carrier protein to load the bacterial antigenic polysaccharides (O1 serotype), which could covalently bind to SpyTag-functionalized nanoparticles AP205 to form nanovaccines. Furthermore, two genes (wbbYandwbbZ) located in the O-antigen biosynthesis gene cluster were knocked out to change the O1 serotype of the engineered strain into the O2 serotype. Both KPO1-SC and KPO2-SC glycoproteins were successfully obtained as expected using our glycoengineering strains. Our work provides new insights into the design of nontraditional bacterial chassis for bioconjugate nanovaccines against infectious diseases.