Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides.
We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.
Fierfort
Nicolas
N
Centre de Recherches sur les Macromolécules Végétales (CERMAV - CNRS), affiliated with Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France.
Samain
Eric
E
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D024861:Genetic Enhancement / Q000379:methods; D007218:Industrial Microbiology / Q000379:methods*; D007785:Lactose / Q000031:analogs & derivatives / Q000378:metabolism; D016254:Mutagenesis, Insertional; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism; D009345:Neisseria meningitidis / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D009844:Oligosaccharides; D012794:Sialic Acids; C010004:N-acetylglucosamine 6-phosphate; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; C000020:N-acetylneuraminoyllactose; D012799:Sialyltransferases; C029156:N-acetyllactosaminide alpha-2,3-sialyltransferase; C018730:N-acetylneuraminate synthase; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D000216:N-Acylneuraminate Cytidylyltransferase; D005634:Fructose-Bisphosphate Aldolase; D007652:Oxo-Acid-Lyases; D002238:Carbohydrate Epimerases; D007785:Lactose; D000117:Acetylglucosamine