Chitin and peptidoglycan fragments are well recognized as pathogen associated molecular patterns (PAMPs). Long-chain oligosaccharides of (1→4)-linked N-acetyl-D-glucosamine (GlcNAc) units indeed activate plants and mammals innate immune system. However, the mechanisms underlying PAMPs perception by lysine motif (LysM) domain receptors remain largely unknown because of insufficient availability of high-affinity molecular probes. Here, we report a two-enzyme cascade to synthesize long-chain (1→4)-linked GlcNAc oligomers.Expression of the D52S mutant of hen egg-white lysozyme (HEWL) in Pichia pastoris at 52 mg.L -1 provided a new glycosynthase catalyzing efficient polymerization of -chitintriosyl fluoride. Selective N-deacetylation at the non-reducing unit of the glycosyl fluoride donor by Sinorhizobium meliloti NodB chitin-N-deacetylase abolished its ability to be polymerized by the glycosynthase but not to be transferred onto an acceptor. Using NodB and D52S HEWL in a one-pot cascade reaction allowed the synthesis on a milligram scale of chitin hexa-, heptaand octasaccharides with yields up to 65% and a perfect control over their size.
During our investigation to find suitable conditions to prepare very high molecular weight partially de‐acetylated hyaluronic acid (HA), we discovered a powerful new method to cleave amide bonds using hydroxylamine salts at neutral pH with remarkable selectivity over common carbamate protecting groups and other carbonyl functionality including unhindered esters.
Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.
Peptidoglycan oligomers constitute precious tools for the biochemical and structural studies of enzymes involved in the bacterial cell wall metabolism. In this study we show that an unprecedented selective Zemplén de-O-acetylation of benzyl chitinbioside peracetate leads to a fast and efficient route to N-acetylmuramyl (1→4) Nacetylglucosaminide disaccharide, a central building block for the synthesis of peptidoglycan oligomers. Starting from known benzyl chitinbioside, NAG-NAM disaccharide pentapeptide is prepared in seven steps with an overall yield of 12%.
Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N‐acetyl‐β‐D‐glucosaminyl‐(1→4)‐1,6‐anhydro‐N‐acetyl‐β‐D‐muramic acid (GlcNAc‐anhMurNAc), a common precursor of Gram‐negative and Gram‐positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram‐positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the in vivo production of Gram‐negative muropeptides containing meso‐diaminopimelic acid, a rarely available amino acid.
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