title: Functional expression of L-fucokinase/guanosine 5'-diphosphate-L-fucose pyrophosphorylase from Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars from exogenous monosaccharides.
abstract: The biosynthesis of glycoconjugates requires the relevant glycosyltransferases and nucleotide sugars that can act as donors. Given the biological importance of posttranslational glycosylation, a facile, robust and cost-effective strategy for the synthesis of nucleotide sugars is highly desirable. In this study, we demonstrate the synthesis of nucleotide sugars from corresponding monosaccharides in a highly efficient manner via metabolic engineering, using an enzymatic approach. This method exploits l-fucokinase/guanosine 5'-diphosphate (GDP)-l-fucose (L-Fuc) pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts l-Fuc into GDP-L-Fuc via an L-Fuc-1-phosphate intermediate. Because L-Fuc and d-arabinose (D-Ara) are structurally similar, it is assumed that the biosynthesis of GDP-D-Ara in a recombinant Saccharomyces cerevisiae strain harboring the FKP gene can occur through a mechanism akin to that of GDP-L-Fuc via the salvage pathway. Thus, we reasoned that by exogenously supplying different monosaccharides structurally related to L-Fuc, it should be possible to produce the corresponding nucleotide sugars with this recombinant yeast strain, regardless of internal acquisition of nucleotide sugars through expression of additive enzymes in the de novo pathway.
authors.affiliation: Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central-2 OSL, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Ta-Wei
authors.initials: TW
authors.lastname: Liu
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Chiba
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kubota
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
mesh_terms: D001441:Bacteroides fragilis / Q000737:chemistry / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods*; D006001:Glycoconjugates / Q000096:biosynthesis*; D006031:Glycosylation; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D007700:Kinetics; D009005:Monosaccharides / Q000378:metabolism; D009711:Nucleotides / Q000096:biosynthesis*; D030781:Organisms, Genetically Modified; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D014170:Transformation, Genetic
chemical_list: D006001:Glycoconjugates; D009005:Monosaccharides; D009711:Nucleotides; D011994:Recombinant Proteins; D006154:Guanosine Diphosphate Fucose; D017853:Phosphotransferases (Alcohol Group Acceptor); C020153:fucokinase