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

• Published in: Carbohydrate research 2010-07, Vol.345 (10), p.1394-1399
• Main Authors: Drouillard, Sophie, Mine, Toshiki, Kajiwara, Hitomi, Yamamoto, Takeshi, Samain, Eric
• Format: Article
• Language: English
• Subjects: 6′-Sialyllactose; Chemical Sciences; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Engineering - methods; Lactose - analogs & derivatives; Lactose - biosynthesis; Magnetic Resonance Spectroscopy; Metabolic engineering; Oligosaccharide; Photobacterium - enzymology; Photobacterium - genetics; Sialyltransferase; Sialyltransferases - genetics; Trisaccharides - biosynthesis
• ISSN: 0008-6215; 1873-426X; 0008-6215
• DOI: 10.1016/j.carres.2010.02.018

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 α-(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 α-(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 α-(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 30 g/L of culture medium.