Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli

• DOI: 10.1007/s10529-016-2151-y
• Publication Date: 2016-06-17T08:55:49Z
• Authors: Hui-Min Qin; Songtao Li; Yu-Fu Zhang; Jian-Wen Wang; Jixuan Li; Shiyi Song; Fuping Lu; Yu Li
• Keywords: 0301 basic medicine; Escherichia coli Proteins; Genetic Vectors; Oligosaccharides; Ketone Oxidoreductases; Lactose; Fucosyltransferases; Nucleotidyltransferases; Biosynthetic Pathways; 3. Good health; 03 medical and health sciences; Multienzyme Complexes; Phosphotransferases (Phosphomutases); Fermentation; Glucokinase; Guanosine Diphosphate Fucose; Escherichia coli; Carbohydrate Dehydrogenases; Transformation, Bacterial; Carbohydrate Epimerases; Mannose; Fucose

To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from D-mannose by two-step fermentation pathway in Escherichia coli.E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using D-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation.Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.