Reassembled Biosynthetic Pathway for Large-Scale Carbohydrate Synthesis:α-Gal Epitope Producing “Superbug”

Abstract

A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galalpha l,3Lac through galactose metabolism has been developed. The plasmid contains a lambda promoter, a c1857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine - Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galalpha 1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 gL(-1)) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5'-diphosphoglucose. This work demonstrates that large-scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.

Enzymatic production of α-d-galactose 1-phosphate by lactose phosphorolysis

“…At the same time the permeabilization procedure allowed diffusion of substrates and products across the cell membrane. A similar result has been reported by Chen et al (2002) who produced a trisaccharide with Triton X-100 permeabilized E. coli cells.…”

Cloning and analysis of the galactose-1-phosphate uridylyltransferase (galt) gene of Gracilariopsis lemaneiformis (Rhodophyta) and correlation between gene expression and agar synthesis

“…Enzymatic activity assay for galactose-1-phosphate uridylyltransferase This was a two-step assay as described by Chen et al (2002) with minor modifications. In brief, GalT-catalyzed reactions were performed at room temperature (24°C) for 20 min in a final volume of 250 μL HEPES buffer (100 mM, pH 7.4) containing 1.6 mM Gal-1-P, 2.8 mM UDP-glucose, and 100 μL of enzyme solution.…”

“Lost sugars” — reality of their biological and medical applications

“…This method has been applied for the production of 3'-sialyllactose [92] as well as the α-gal epitope [93]. The Wang group introduced a "superbug" for α-gal epitope synthesis, i.e., a metabolically engineered E. coli strain containing a plasmid that harbours a gene cluster encoding all enzymes in the biosynthetic pathway [94]. The main advantage of these whole-cell methods is the utilisation of the microorganisms' own metabolic pathways for synthesis from inexpensive materials without the addition of nucleotides or the need for complicated enzyme purification [90].…”