The Construction of E.coli Engineering Strain for Sialyllactose Production

Abstract

Abstract: Free sialylated oligosaccharides are known to have anti-infective and immunostimulating properties and also known to promote bifidobacterium proliferation, thus investigating the microbial synthetic route of sialylated oligosaccharides is of great value. Biosynthesis of sialyllactose involves N-acetylglucosamine-6-phosphate-epimerase（neuC）, sialic acid synthase（neuB）, CMP-Neu5Ac synthetase（neuA）and α-2, 3-sialyltransferase（nst）. We engineered a biosynthetic pathway sialyllactose production in E.coli JM109, using the expression vector pSTV29, by coexpressing the α-2, 3-sialyltransferase gene from Neisseria meningitidis with the neuA, neuB and neuC Campylobacter jejuni genes encoding CMP-NeuAc synthetase, sialic acid synthase and N-acetylglucosamine-6-phosphate-epimerase, respectively. Under the optimized fermentation conditions（inoculum volume 2%, 10g/L lactose, fermentation volume 50 mL/250 mL, temperature 34℃, rotation speed 180 r/min, fermentation time 30 h）, sialyllactose of 2.45 g/L was obtained. The above results provide a platform for exploring commercial production of sialylated oligosaccharides and its functional analogues in heterogeneous microbial hosts.

Key words: Sialyllactose, E.coli, Metabolic engineering

Jin Wenbin, Zhang Xiaoxiao, Li Yu, Lu Fuping. The Construction of E.coli Engineering Strain for Sialyllactose Production[J]. Biotechnology Bulletin, 2014, 0(10): 201-206.

References

[1] E, Giuliani F, Occhi L, et al. Benefits of donor human milk for preterm infants：current evidence[J]. Early Human Development, 2009, 85（10）：S9-S10.
[2] F. Recent studies on reaction pathways and applications of sugar orthoesters in synthesis of oligosaccharides[J]. Carbohydrate Research, 2007, 342（3）：345-373.
[3] B, Thurl S, Zeng JR, et al. Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spec-trometry[J]. Analytical Biochemistry, 1994, 223（2）：218-226.
[4] T, Katayama S, Kajiwara H, et al. An α2, 6-sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both sialyltransferase and neuraminidase activity[J]. Glycobiology, 2010, 20（2）：158-165.
[5] WF, Tavarez JJ, Crowley J, et al. Purification and characterization of the Escherichia coli Kl neuB gene product N-acetylneuraminic acid synthetase[J]. Glycobiology, 1997, 7（5）：697-701.
[6] WF, Daines DA, Murkin AS, et al. The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase[J]. Journal of Bacteriology, 2004, 186（3）：706-712.
[7] DA, Wright LF, Chaffin DO, et al. NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1[J]. FEMS Microbiology Letters, 2000, 189（2）：281-284.
[8] X, Kiechle FL. Glycosphingolipids in health and disease[J]. Annals of Clinical & Laboratory Science, 2004, 34（1）：3-13.
[9] E, Drouillard S, Heyraud A, et al. Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli[J]. Carbohydrate Research, 1997, 302（1）：35-42.
[10] S, Samain E. Genetic engineering of Escherichia coli for the production of N I, N II-diacetylchitobiose（chitinbiose）and its utilization as a primer for the synthesis of complex carbohydrates[J]. Metabolic Engineering, 2005, 7（4）：311-317.
[11] J, Honda Y, Nagae M, et al. 1, 2-α-L-Fucosynthase：a glycosynthase derived from an inverting α-glycosidase with an unusual reaction mechanism[J]. FEBS Letters, 2008, 582（27）：3739-3743.
[12] T, Heyraud A, Bosso C, et al. Highly efficient biosynthesis of the oligosaccharide moiety of the GD3 ganglioside by using metabolically engineered Escherichia coli[J]. Angewandte Chemie, 2005, 117（9）：1374-1376.
[13] S, Kornfeld R, Neufeld EF, et al. The feedback control of sugar nucleotide biosynthesis in liver[J]. Proceedings of the National Academy of Sciences of the United States of America, 1964, 52（2）：371.
[14] N, Samain E. Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides[J]. Journal of Biotechnology, 2008, 134（3）：261-265.
[15] S, Mine T, Kajiwara H, et al. Efficient synthesis of 6'-sialyllactose, 6, 6'-disialyllactose, and 6'-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224[J]. Carbohydrate Research, 2010, 345（10）：1394-1399.