Abstract
Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitides, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and α-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an α-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce α-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Varki, A. 1993. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3: 97–130.
Harduin-Lepers, A., Recchi, M.-A. and Delannoy, P. 1995. 1994, the year of sialyltransferases. Glycobiology 5: 741–758.
Tsuji, S., Datta, A.K., and Paulson, J.C. 1996. Systematic nomenclature for sialyltransferases. Glycobiology 6: v–vii.
Weisgerber, C., Hansen, A., and Frosch, M. 1991. Complete nucleotide and deduced protein sequence of CMP-NeuAc: poly-alpha-2,8 sialosyl sialyltransferase of Escherichia coliK1. Glycobiology 1: 357–365.
Frosch, M., Edwards, U., Bousset, K., Krausse, B. and Weisgerber, C. 1991. Evidence for a common molecular origin of the capsule gene loci in Gram-negative bacteria expressing group II capsular polysaccharides. Mol. Microbiol. 5: 1251–1263.
Gilbert, M., Watson, D.C., Cunningham, A.-M., Jennings, M.P., Young, N.M., and Wakarchuk, W.W. 1996. Cloning of the lipooligosaccharide α-2,3-sialyHransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae. J. Biol. Chem. 271: 28271–28276
Moran, A.P., Prendergast, M.M., and Appelmelk, B.J. 1996. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol. Med. Microbiol. 16: 105–115.
Kajihara, Y., Yamamoto, T., Nagae, H., Nakashizuka, M., Sakakibara, T., and Terada, I. 1996. A novel α-2,6-sialyltransferase: transfer of sialic acid to fucosyl and sialyl trisaccharides. Journal of Organic Chemistry 61: 8632–8635.
Gilbert, M., Cunningham, A.-M., Watson, D.C., Martin, A., Richards, J.C., and Wakarchuk, W.W. 1997. Characterization of a recombinant Neisseria meningitidisα-2,3-sialyttransferaseand its acceptor specificity. Eur. J. Biochem. 249: 187–194.
Gilbert, M., Watson, D.C., and Wakarchuk, W.W. 1997. Purification and characterization of the recombinant CMP-sialic acid synthetase from Neisseria meningitidis. Biotechnology Letters 19: 417–420.
Warren, L. and Blacklow, R.S. 1962. The biosynthesis of cytidine 5′-monophospho-N-acetylneuraminic acid by an enzyme from Neisseria meningitidis. J. Biol. Chem. 237: 3527–3534.
Ichikawa, Y., Shen, G.-J., and Wong, C.-H. 1991. Enzyme-catalyzed synthesis of sialyl oligosaccharide with in situregeneration of CMP-sialic acid. Journal of the American Chemical Society 113: 4698–4700.
Hannig, G. and Makrides, S.C. 1998. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol. 16: 54–60.
Netzer, W.J. and Hartl, F.U. 1997. Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature 388: 343–349.
Schurig, H., Beaucamp, N., Ostendorp, R., Jaenicke, R., Adler, E., and Knowles, J.R. 1995. Phosphoglycerate kinase and triosephosphate isomerase from the hyperther-mophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex. EMBO J. 14: 442–151.
Wakarchuk, W.W., Martin, A., Jennings, M.R., Moxon, E.R., and Richards, J.C. 1996. Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J. Biol. Chem. 271:19166–19173.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gilbert, M., Bayer, R., Cunningham, AM. et al. The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion. Nat Biotechnol 16, 769–772 (1998). https://doi.org/10.1038/nbt0898-769
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0898-769
- Springer Nature America, Inc.