Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1.
We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.
Yavuz
Elif
E
Centre de Recherches sur les Macromolécules Végétales, 601 rue de la Chimie, BP 53X, 38041 Grenoble, cedex 09, France.
Drouillard
Sophie
S
Samain
Eric
E
Roberts
Ian
I
Priem
Bernard
B
D000818:Animals; D018959:CD57 Antigens / Q000096:biosynthesis / Q000737:chemistry*; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D015202:Protein Engineering; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000378:metabolism
D018959:CD57 Antigens; D029968:Escherichia coli Proteins; D009844:Oligosaccharides; C097522:KfiD protein, E coli; D014533:Uridine Diphosphate Glucose Dehydrogenase; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase; D005966:Glucuronidase