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Applied and Environmental Microbiology, April 2003, p. 2110-2115, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2110-2115.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

P1 Trisaccharide (Gal1,4Galß1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli

Ziye Liu,^1,2 Yuquan Lu,¹ Jianbo Zhang,¹ Keith Pardee,² and Peng George Wang^1*

Department of Chemistry, Wayne State University, Detroit, Michigan 48202,¹ Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada²

Received 3 July 2002/ Accepted 13 November 2002

The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Gal1,4Galß1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori ß-l,4-galactosyltransferase and a Neisseria meningitidis -1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.

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* Corresponding author. Mailing address: Department of Chemistry, Wayne State University, Detroit, MI 48202. Phone: (313) 993-6759. Fax: (313) 577-2554. E-mail: pwang{at}chem.wayne.edu.

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Applied and Environmental Microbiology, April 2003, p. 2110-2115, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2110-2115.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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