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.

[1]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  F. Kiechle,et al.  Glycosphingolipids in Health and Disease , 2004 .

[3]  B. Priem,et al.  Large‐Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli , 2003, ChemBioChem.

[4]  I. Werner,et al.  On the presence of sialic acid in certain glycoproteins and in gangliosides. , 1952, Acta Societatis Medicorum Upsaliensis.

[5]  E. Samain,et al.  Highly efficient biosynthesis of the oligosaccharide moiety of the GD3 ganglioside by using metabolically engineered Escherichia coli. , 2005, Angewandte Chemie.

[6]  Y. Tsukada,et al.  Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase. , 1998, Carbohydrate research.

[7]  B. Priem,et al.  A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. , 2002, Glycobiology.

[8]  S. Cottaz,et al.  Genetic engineering of Escherichia coli for the production of NI,NII-diacetylchitobiose (chitinbiose) and its utilization as a primer for the synthesis of complex carbohydrates. , 2005, Metabolic engineering.

[9]  C. Dumon,et al.  Production of Lewis x Tetrasaccharides by Metabolically Engineered Escherichia coli , 2006, Chembiochem : a European journal of chemical biology.

[10]  A. Ozaki,et al.  Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling , 2000, Applied Microbiology and Biotechnology.

[11]  E. Vimr,et al.  Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase. , 1997, Glycobiology.

[12]  B. Washburn,et al.  New method for generating deletions and gene replacements in Escherichia coli , 1989, Journal of bacteriology.

[13]  A. S. Murkin,et al.  The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase , 2004, Journal of bacteriology.

[14]  L. Juneja,et al.  Preparation ofN-acetylneuraminic acid from delipidated egg yolk , 1992, Glycoconjugate Journal.

[15]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[16]  A. Varki,et al.  Biological roles of oligosaccharides: all of the theories are correct , 1993, Glycobiology.

[17]  S. Kornfeld,et al.  THE FEEDBACK CONTROL OF SUGAR NUCLEOTIDE BIOSYNTHESIS IN LIVER. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Byung-Gee Kim,et al.  Production of N-acetylneuraminic acid from N-acetylglucosamine and pyruvate using recombinant human renin binding protein and sialic acid aldolase in one pot , 2004 .

[19]  Bing Wang,et al.  Brain ganglioside and glycoprotein sialic acid in breastfed compared with formula-fed infants. , 2003, The American journal of clinical nutrition.

[20]  Yinghong Gao,et al.  The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion , 1998, Nature Biotechnology.

[21]  B. Stahl,et al.  Oligosaccharides from milk. , 2007, The Journal of nutrition.