Production of Lewis x Tetrasaccharides by Metabolically Engineered Escherichia coli

Abstract

Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram-scale by high-cell-density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for alpha-3 fucosyltransferase and the Neisseria meningitidis lgtB gene for beta-4 galactosyltransferase. The first compound Galbeta-4(Fucalpha-3)GlcNAcbeta-4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin-synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galbeta-4(Fucalpha-3)GlcNAcbeta-3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for beta-3 N-acetylglucosaminyltransferase.

Exploiting Bacterial Glycosylation Machineries for the Synthesis of a Lewis Antigen-containing Glycoprotein

“…Fucosylation of free oligosaccharides with LacNAc-epitopes for the assembly of the Le x antigen was previously accomplished in live E. coli cells using H. pylori fucosyltransferases (32). The design of the glycoengineering approach followed here, however, imposes that the glycans must be built onto the UndPP carrier to allow en bloc transfer to acceptor proteins.…”

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

“…However, many proteins, particularly mammalian proteins, do not express well in a useful form in E. coli due to the absence of a proper folding environment or post translational modification. For the latter a new strategy, confirmed by successful production of glycosylated protein, has been proposed to obtain certain posttranslational protein modification in E. coli (2)(3)(4).…”

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains

“…Glycosyltransferases generally have strict regio-and acceptor specificity and are therefore good catalysts for defined oligosaccharide syntheses. Efficient preparations of Le x and H antigen oligosaccharides using ␣-1,3-and ␣-1,2-fucosyltransferases from H. pylori have been reported (11)(12)(13). However, as described in the literature (11), the use of glycosyltransferases requires an expensive sugar nucleotide or a complex system for nucleotide recycling.…”