Glycosynthase-Based Synthesis of Peptidoglycan Oligosaccharides to Address Bacterial Cell-Wall Elongation Processes

Most antibiotics used in human medicine inhibit the biosynthesis of peptidoglycan (PG), which is a vital component of the bacterial cell wall. However, the rapid rise in antimicrobial resistance (AMR) represents a major global health threat. Gaining deeper insight into PG metabolism is a crucial step in combating AMR, an objective achievable only through the use of well-defined molecular probes. In this study, we developed a chemo-enzymatic method to synthesize PG oligosaccharides using a lysozyme-derived glycosynthase. To prevent unwanted polymerization and enable precise control over the product size, a glycosyl fluoride donor bearing a terminal galactosyl unit was designed. The trisaccharide Gal-β-(1→4)-GlcNAc-β-(1→4)-1,6-anhydro-MurNAc was biosynthesized in metabolically engineered <i>Escherichia coli</i> cells and subsequently fluorinated at the reducing end via chemical modification. Successive glycosylation and degalactosylation steps using this donor, starting from two disaccharide acceptors, led to the synthesis of PG tetra-, hexa-, and octasaccharides, with a 60-70% yield at each cycle. These compounds were used to probe the specificity of <i>E. coli</i> DedD, a SPOR-domain-containing protein involved in bacterial cell division. NMR spectroscopy and NMR-restraint-driven molecular dynamics provide new insights into the role of this protein relative to those of other <i>E. coli</i> SPOR domain-containing proteins.