Glycoconjugate vaccines are important for prevention of bacterial disease, especiallyamid rising antibiotic resistance. The conventional conjugation methods, though effective, suffer from drawbacks like uncontrolled coupling of glycan and heterogeneous immunogenicity. Protein glycan coupling technology (PGCT) offers site-specific glycosylation through bacterial oligosaccharyltransferases (OSTs). The widely used Campylobacter jejuni PglB (CjPglB), has strict specificity for glycans with a reducing-end N-acetylhexosamine, resulting in low glycosylation efficiency of some vaccine-relevant O-antigen glycan structures. The research of the present study focuses on improvement of glycan-protein conjugation by screening CjPglB homologues with higher transfer activity toward O-polysaccharide (O-PS) antigens from Francisella tularensis strain Schu S4 and enterotoxigenic Escherichia coli (ETEC) serotype O78 for preparation of glycoconjugate vaccines against more O-antigens. The objective of this research study is to identify OST homologues with equal or greater transfer efficiency of vaccine-relevant O-PS antigens to immunogenic carrier proteins such as protein D (PD) from Haemophilus influenzae, with the goal of extending the spectrum of bacterial O-PS antigens that can be conjugated to vaccine carrier proteins. O-PS antigen biosynthetic genes from F. tularensis Schu S4 and ETEC O78 were cloned and expressed in a laboratory strain of E. coli (strain CLM24) to generate novel glycoconjugates. We anticipate that enrichment of glycosylated PD will enhance vaccine immunogenicity, with greater O-PS antigen-specific IgG titers. Using this approach helps us to quickly determine the most appropriate OST for a target O-antigen, thereby greatly improving the effectiveness, specificity, and scale of PGCT-based conjugate vaccine manufacturing.