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.
We have previously reported the efficient conversion of lactose into 3'-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for alpha-(2-->3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6'-sialyllactose with a strain expressing alpha-(2-->6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that alpha-(2-->6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6'-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6'-disialyllactose, was also produced. Formation of 6,6'-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6'-sialyllactose was almost the only product detected and its final concentration was higher than 30g/L of culture medium.
Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), BP53, 38041 Grenoble cedex 9, France.
Sophie
S
Drouillard
Toshiki
T
Mine
Hitomi
H
Kajiwara
Takeshi
T
Yamamoto
Eric
E
Samain
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D007785:Lactose / Q000031:analogs & derivatives / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D010776:Photobacterium / Q000201:enzymology* / Q000235:genetics; D012799:Sialyltransferases / Q000235:genetics*; D014312:Trisaccharides / Q000096:biosynthesis
C553092:2-keto-3-deoxy-manno-octonyllactose; C403777:6'-sialyllactose; D014312:Trisaccharides; C509062:disialyllactose; D012799:Sialyltransferases; D007785:Lactose