Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria

A large-scale production system of uridine 5'-diphospho-galactose (UDP-Gal) has been established by the combination of recombinant Escherichia coli and Corynebacterium ammoniagenes. Recombinant E. coli that overexpress the UDP-Gal biosynthetic genes galT, galK, and galU were generated. C. ammonia-genes contribute the producion of uridine triphosphate (UTP), a substrate for UDP-Gal biosynthesis, from orotic acid, an inexpensive precursor of UTP. UDP-Gal accumulated to 72 mM (44 g/L) after a 21 h reaction starting with orotic acid and galactose. When E. coli cells that expressed the 1,4-galactosyltrans-ferase gene of Neisseria gonorrhoeae were coupled with this UDP-Gal production system, 372 mM (188 g/L) globotriose (Gal1 -4Gal1 -4Glc), a trisaccharide portion of verotoxin receptor, was produced after a 36 h reaction starting with orotic acid, galactose, and lactose. No oligosaccharide by-products were observed in the reaction mixture. The production of globotriose was several times higher than that of UDP-Gal. The strategy of producing sugar nucleotides by combining metabolically engineered recombinant E. coli with a nucleoside 5'-triphosphate producing microorganism, and the concept of producing oligosaccharides by coupling sugar nucleotide production systems with glycosyltransferases, can be applied to the manufacture of other sugar nucleotides and oligosaccharides.

1. Gabius, H.-J. and Gabius, S. 1997. Glycoscience: status and perspectives. Chapman and Hall GmbH, Weinheim, Germany.
2. Khan, S.H. and O'Neill, R.A. 1996. Modern methods in carbohydrate synthesis. Harwood Academic Publishers, Amsterdam, The Netherlands.
3. Leloir, L.F. 1971. Two decades of research on the biosynthesis of saccharides. Science 172: 1299−1303. | PubMed  | ISI | ChemPort |
4. Gilbert, M., Watson, D.C., Cunningham, A.-M., Jennings, M.R., Young, N.M., and Wakarchuk, W.W. 1996. Cloning of the lipooligosaccharide -2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae. J. Biol. Chem. 271: 28271−28276. | Article | PubMed  | ISI | ChemPort |
5. Kolkman, M.A.B., Wakarchuk, W., Nuijten, P.J.T., van der Zeijst, B.A.M. 1997. Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit. Mol. Microbiol. 26: 197−208. | Article | PubMed  | ISI | ChemPort |
6. Martin, S.L., Edbrooke, M.R., Hodgman, T.C., van den Eijnden, D.H., and Bird, M.I. 1997. Lewis X biosynthesis in Helicobacter pylori: molecular cloning of an (1,3)-fucosyltransferase gene. J. Biol. Chem. 272: 21349−21356. | Article | PubMed  | ISI | ChemPort |
7. Ge, Z., Chan, N.W.C., Palcic, M.M. and Taylor, D.E. 1997. Cloning and heterologous expression of an -1,3-fucosyltransferase gene from the gastric pathogen Helicobacter pylori. J. Biol. Chem. 272: 21357−21363. | Article | PubMed  | ISI | ChemPort |
8. Gotschlich, E.C. 1998. Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them. US 5,705,367.
9. Yamamoto, T., Nakashizuka, M., and Terada, I. 1998. Cloning and expression of a marine bacterial -galactoside 2,6-sialyltransferase gene from Photobacterium damsela JT0160. J. Biochem. 123: 94−100. | PubMed  | ISI | ChemPort |
10. Wong, C.-H., Haynie, S.L., and Whitesides, G.M. 1982. Enzyme-catalyzed synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose. J. Org. Chem. 47: 5416−5418. | ISI | ChemPort |
11. Wong, C.-H., Wang, R., and Ichikawa, Y. 1992. Regeneration of sugarnucleotide for enzymatic oligosaccharide synthesis: use of gal-1-phosphate uridyltransferase in the regeneration of UDP-galactose, UDP-2-deoxygalactose, and UDP-galactosamine. J. Org. Chem. 57: 4343−4344. | ISI | ChemPort |
12. Tochikura, T., Mugibayashi, Y., Kawai, H., Kawaguchi, K., and Ogata, K. 1970. Studies on microbial metabolism of sugar nucleotides Part IV. Effect of water content of dried cells of Torulopsis Candida on UDP-hexose fermentation. Amino Acid and Nucleic Acid 22: 144−150.
13. Lingwood, C.A., Law, H., Richardson, S., Petric, M., Brunton, J.L., Grandis, S.D. et al. 1987. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J. Biol. Chem. 262: 8834−8839. | PubMed  | ISI | ChemPort |
14. Lee, Y.C. 1990. High-performance anion-exchange chromatography for carbohydrate analysis. Anal. Biochem. 189: 151−162. | Article | PubMed  | ISI | ChemPort |
15. Teshiba, S. and Furuya, A. 1988. Production of nucleotides and nucleosides by fermentation. Gordon and Breach Science Publishers, New York.
16. Fujio, T. and Maruyama, A. 1997. Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (part I). Biosci. Biotech. Biochem. 61: 956−959. | ISI | ChemPort |
17. Whistler, R.L. and Durso, D.F. 1950. Chromatographic separation of sugars on charcoal. J. Am. Chem. Soc. 72: 677−679. | ISI | ChemPort |
18. Navarro, A., Caruel, H. Rigal, L., and Phemius, R 1997. Continuous Chromatographic separation process: simulated moving bed allowing simultaneous withdrawal of three fractions. J. Chromatogr. 770: 39−50. | Article | ISI | ChemPort |
19. Sambrook, J., Fritsh, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
20. Fujio, T., Nishi, T., Ito, S., and Maruyama, A. 1997. High level expression of XMP aminase in Escherichia coli and its application for the industrial production of 5'-guanylic acid. Biosci. Biotech. Biochem. 61: 840−845. | ISI | ChemPort |
21. Lemaire, H.-G. and Müller-Hill, B. 1986. Nucleotide sequeces of the galE gene and the galT gene of E. coli. Nucleic Acids Res. 14: 7705−7711. | PubMed  | ISI | ChemPort |
22. Debouck, C., Riccio, A., Schumperli, D., McKenney, K., Jeffers, J., Hughes, C. et al. 1985. Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon. Nucleic Acids Res. 13: 1841−1853. | PubMed  | ISI | ChemPort |
23. Weissborn, A.C., Liu, Q., Rumley, M.K., and Kennedy, E.P. 1994. DTP: -D-glu-cose-1-phosphate uridyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme. J. Bacteriol. 176: 2611−2618. | PubMed  | ISI | ChemPort |
24. Lahti, R., Pitkäranta, T., Valve, E., llta, I., Kukko-Kalske, E., and Heinonen, J. 1988. Cloning and characterization of the gene encoding inorganic pyrophos-phatase of Escherichia coli K-12. J. Bacteriol. 170: 5901−5907. | PubMed  | ISI | ChemPort |
25. Lagunas, R. and Díez-Masa, J.C. 1994. Separation and analysis of 4'-epimeric UDP-sugars by ion-pair reverse-phase HPLC. Anal. Biochem. 216: 188−194. | Article | PubMed  | ISI | ChemPort |

	Top

	Top