Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.