title: Efficient chemoenzymatic synthesis of uridine 5'-diphosphate N-acetylglucosamine and uridine 5'-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes.
abstract: Uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5'-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.
authors.affiliation: a Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University , Hangzhou , P. R. China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.affiliation: a Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University , Hangzhou , P. R. China.
authors.forename: Chen
authors.initials: C
authors.lastname: Qi
authors.affiliation: b School of Biological and Chemical Engineering , Zhejiang University of Science & Technology , Hangzhou , P. R. China.
authors.forename: Peilian
authors.initials: P
authors.lastname: Wei
authors.affiliation: a Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University , Hangzhou , P. R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: a Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University , Hangzhou , P. R. China.
authors.forename: Jin
authors.initials: J
authors.lastname: Cai
authors.affiliation: a Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University , Hangzhou , P. R. China.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D016983:Enterococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D043564:Inorganic Pyrophosphatase / Q000235:genetics / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C114468:uridine 5'-(2-trifluoroacetamido-2-deoxyglucopyranosyl diphosphate); D014537:Uridine Diphosphate N-Acetylglucosamine; D014530:Uridine Diphosphate; D010770:Phosphotransferases; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D043564:Inorganic Pyrophosphatase; D000117:Acetylglucosamine
keywords: Biosynthesis; N-acetylglucosamine-1-phosphate uridyltransferase; N-acetylhexosamine 1-kinase; one-pot system; uridine 5′-diphosphate N-acetylglucosamine; uridine 5′-diphosphate N-trifluoacetyl glucosamine