title: In-microbe formation of nucleotide sugars in engineered Escherichia coli.
abstract: 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 μg/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-XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP-galacturonic acid (UDP-GalA) and UDP-galactose (UDP-Gal). ¹³C- and ¹⁵N-labeled NDP-sugars are formed using [¹³C] glucose as the carbon source and with [¹⁵N]NH₄Cl as the nitrogen source.
authors.affiliation: Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA 30602, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Yang
authors.forename: Yael
authors.initials: Y
authors.lastname: Bar-Peled
authors.forename: James Amor
authors.initials: JA
authors.lastname: Smith
authors.forename: John
authors.initials: J
authors.lastname: Glushka
authors.forename: Maor
authors.initials: M
authors.lastname: Bar-Peled
mesh_terms: D001483:Base Sequence; D050260:Carbohydrate Metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D009711:Nucleotides / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009711:Nucleotides; D014539:Uridine Diphosphate Sugars