Journal
ANALYTICAL BIOCHEMISTRY
Volume 421, Issue 2, Pages 691-698Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.12.028
Keywords
Nucleotide sugar metabolism; UDP-GalA; UDP-Gal; UDP-GlcNAcA; UDP-XylNAc; UDP-Glc; C-13-labeled nucleotide sugar; N-15-labeled nucleotide sugar; E. coli; In-microbe
Funding
- National Science Foundation [IOB-0453664]
- BioEnergy Science Center
- Office of Biological and Environmental Research in the Department of Energy's Office of Science
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Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated in-microbe, reactions occur within 2 to 4 h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5 mu g/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP-2-acetamido-2-deoxy-glucuronic acid (UDP-GlcNAcA) and UDP-2-acetamido-2-deoxy-xylose (UDP-Xyl-NAc). Using similar strategies, we also engineered E. coli to synthesize UDP-galacturonic acid (UDP-GalA) and UDP-galactose (UDP-Gal). C-13- and N-15-labeled NDP-sugars are formed using [C-13] glucose as the carbon source and with [N-15]NH4Cl as the nitrogen source. (C) 2011 Elsevier Inc. All rights reserved.
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