Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli.
OBJECTIVE
To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from D-mannose by two-step fermentation pathway in Escherichia coli.


RESULTS
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.


CONCLUSIONS
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.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Hui-Min
HM
Qin
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Songtao
S
Li
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Yu-Fu
YF
Zhang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Jian-Wen
JW
Wang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Jixuan
J
Li
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Shiyi
S
Song
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
Fuping
F
Lu
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China. liyu@tust.edu.cn.|Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China. liyu@tust.edu.cn.|National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China. liyu@tust.edu.cn.
Yu
Y
Li
D053898:Biosynthetic Pathways; D002237:Carbohydrate Dehydrogenases / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000235:genetics; D005285:Fermentation; D005643:Fucose / Q000737:chemistry*; D005647:Fucosyltransferases / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005941:Glucokinase / Q000235:genetics; D006154:Guanosine Diphosphate Fucose / Q000737:chemistry; D007658:Ketone Oxidoreductases / Q000235:genetics; D007785:Lactose / Q000737:chemistry; D008358:Mannose / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics; D014169:Transformation, Bacterial
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009844:Oligosaccharides; C110682:wcaG protein, E coli; D006154:Guanosine Diphosphate Fucose; D005643:Fucose; D002237:Carbohydrate Dehydrogenases; D007658:Ketone Oxidoreductases; D005647:Fucosyltransferases; D005941:Glucokinase; D009713:Nucleotidyltransferases; C021546:mannose-1-phosphate guanylyltransferase (GDP); D002238:Carbohydrate Epimerases; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase; D007785:Lactose; D008358:Mannose
Enzymatic synthesis; Fermentation; Fucosyloligosaccharide; GDP-fucose; Human milk oligosaccharides; Mannose; Oligosaccharides