“Peptidoglycan (PG) is a complex biopolymer made of polysaccharide chains which are cross-linked by peptide bridges. Glycan chains are formed by the repetition of dimeric units made of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by β(1→4) bonds. PG is an essential and specific component of the bacterial cell wall which is needed to confer osmotic stability and morphological robustness to the cell throughout the bacterial life cycle. Any defect during the cell wall assembly is harmful, indeed lethal for the bacteria. Therefore, given that PG is not found in mammals, most antibiotics used in human medicine target peptidoglycan biosynthesis enzymes and prevent bacterial growth. However, the excessive use of antibiotics and lack of new active molecules have led to the emergence and spread of resistant bacteria. For many decades, antibiotic resistance has been rising to dangerously high levels all over the world. It is estimated to be responsible for 700,000 deaths a year and unless action is taken, this figure could rise to 10 million by 2050. This projection would make antibiotic resistance a bigger threat than cancer. As major antibiotic resistance mechanisms are associated with mutations in enzymes involved in the PG metabolism, in-depth structural and functional analyses of these key biocatalysts are essential to develop new antibiotics. This work requires the use of substrates or molecular probes with perfectly defined structures. The synthesis of PG oligomers is a complex task and to date, only few groups worldwide have addressed this work, mainly by chemical approaches requiring numerous steps.
In this context, this PhD thesis aimed at developing innovative chemo-enzymatic and microbiological methodologies to produce PG oligomers in quantities allowing biochemical and structural studies. First, Hen egg-white lysozyme, a well-known peptidoglycan-degrading enzyme has been converted into a glycosynthase and this new enzymatic tool has enabled the oligomerization of the PG disaccharide motif. In the meantime, we have developed two new straightforward accesses to the GlcNAc-MurNAc precursor. The first one is a chemical approach based on the regioselective modification of chitinbiose. The second one relies on the metabolic engineering of the peptidoglycan recycling pathway of Escherichia coli in order to produce the disaccharide in vivo. Finally, by using the glycosynthase and the precursor, we have implemented a chemo-enzymatic synthesis of PG oligomers up to the octamer with perfect control over their size. These molecules are currently used to carry out functional and structural analyses of the bacterial cell wall enzymatic machinery.
Key words: peptidoglycan oligomers, glycosynthase, metabolic engineering, chemo-enzymatic synthesis, antibiotic resistance.”