Production of UDP-Gal

A new industrial avenue to oligosaccharide synthesis may be opened by an approach using whole-cell biotransformations. Researchers from Kyowa Hakko Co. (Japan) have developed systems for the large-scale production of UDP-Gal and globotriose (Fig. 11) from inexpen-... 

Figure 11. Production of UDP-Gal and globotriose using metabolically engineered microbial mixed cultures.

Scheme 5.7 Combination of three bacterial strains for the production of globotriose (13) with regeneration of UDP-Gal [40]. Combination of C. ammoniagenes DN510 (A), E. coli NM 522 pNT25/pNT32 (B) and E. coli NM522 pGT5 (C).

The first prominent example of large-scale oligosaccharide production by bacterial coupling was described by Koizumi et al. (Scheme 5.7) [40]. Three engineered bacterial strains were combined for the production of globotriose (13) including regeneration of UDP-Gal. 

The reaction mixture contained N-acetylglucosamine, UDP-Gal, MnCh, and buffer at pH 8.0. The reaction was started by the addition of the enzyme. Samples were transferred at intervals to cacodylate buffer on ice (pH 6.5) to terminate the reaction. Samples (10 /u.L) were analyzed by HPLC. The conversion of UDP-Gal to UDP is shown in Figure 9.68. Each panel represents a different time point, from 0 to 60 minutes. During the incubation, the disappearance of the substrate and the formation of the two products is seen. The enzyme was obtained from commercial sources and human serum. 

Figure 4 The fusion of epimerase (UDP-Gal-4-epimerase) and al,3GalT (bovine al,3 galactosyltransferase-EC 2.4.1.151) enables the use of UDP-Glc instead of UDP-Gal, a 40-fold reduction in the cost of production of the a-Gal epitopes.

Scheme 11 Production of superbeads and biosynthetic pathway of globotriose with regeneration of UDP-Gal.

Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100°C and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the en me reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (l- )-P-D-galactan. The supernatant was analysed for soluble labelled products. 

CMP-NeuAc synthetase (EC 2.7.7.43) to produce CMP-NeuAc. The by-product pyrophosphate (PPi) is hydrolyzed to phosphate (Pi) by inorganic pyrophosphatase (PPase). Sialyla-tion is accomplished with a2,3-sialyltransferase (< 2,3NeuAcT) or a2,6-sialyltransferase (a2,3NeuAcT), respectively. The released CMP is again converted to CDP, to CTP, and finally to CMP-NeuAc. The UDP-Gal and CMP-NeuAc regeneration schemes have been combined in a one-pot reaction and applied to the synthesis of sialyl Lewis X. 

Most assays for this activity either determine the amount of radioactive product formed from radioactive UDP-Gal or measure the amount of UDP present using a coupled assay in which the UDP formed is coupled to the formation of NAD or NADH. 

Figure 2 Examples of glycosyl donors. Nucleotide donors are represented by UDP-galactose (UDP-Gal), GDP-fucose (GDP-Fuc), GMP-N-acetyIneuraminic acid (CMP-sialic acid, CMP-Neu5Ac), GDP-galactose (GDP-Gal), dTDP-rhamnose (dTDP-Rha), and dTDP-daunosamine lipid phosphate donors are represented by Lipid II and dolichol-phosphate-glucose (Dol-P-GIc) sugar phosphate donors are represented by glucose-1-phosphate. The saccharides are transferred from these donors by GTs to form oligo/polysaccharides and glycoconjugate products.

---