Cellular production of glycosylated products

A metabolically engineered cell for producing a glycosylated product comprising a disaccharide or oligosaccharide that comprises at least two different monosaccharide subunits selected from the group consisting of a mammalian milk di- or oligosaccharide, a human milk di- or oligosaccharide, 0-antigen, enterobacterial common antigen (ECA), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), and antigens of the human ABO blood group system, wherein the cell:

• (i) is capable of expressing a variant yeast or fungal glutamine:fructose-6-phosphate aminotransferase that differs from SEQ ID NO:1 by a V12L, a Q96H, a Q157R and/or an E343V mutation and having glutamine:fructose-6-phosphate aminotransferase activity, and
• (ii) is capable of synthesizing UDP-N-acetylglucosamine (UDP-GlcNAc), and
• (iii) is capable of expressing a glycosyltransferase,
• wherein the cell utilizes UDP-GlcNAc to produce the glycosylated product.

A vector comprising an isolated nucleic acid molecule encoding a variant yeast or fungal glutamine:fructose-6-phosphate aminotransferase, wherein the variant yeast or fungal glutamine:fructose-6-phosphate aminotransferase is a protein having glutamine:fructose-6-phosphate aminotransferase activity and that differs from SEQ ID NO:

• 1 by a V12L, a Q96H, a Q157R and/or an E343V mutation and that:
• (i) comprises the polypeptide sequence of any one of SEQ ID NOs:
• 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 that, or
• (ii) comprises an amino acid sequence that is at least 80.0% sequence identical over a stretch of at least 200 amino acid residues to the amino acid sequence of any one of SEQ ID NOs:
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, respectively.

A method of using an isolated nucleic acid molecule encoding a variant yeast or fungal glutamine:fructose-6-phosphate aminotransferase, wherein the variant yeast or fungal glutamine:fructose-6-phosphate aminotransferase is a protein having glutamine:fructose-6-phosphate aminotransferase activity and that differs from SEQ ID NO:

• 1 by a V12L, a Q96H, a Q157R and/or an E343V mutation and that:
• (i) comprises the polypeptide sequence of any one of SEQ ID NOs:
• 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 that, or
• (ii) comprises an amino acid sequence that is at least 80.0% sequence identical over a stretch of at least 200 amino acid residues to the amino acid sequence of any one of SEQ ID NOs:
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, respectively,
• for producing a glycosylated product that is derived from UDP-GlcNAc and comprises a disaccharide or oligosaccharide that comprises at least two different monosaccharide subunits and that is selected from the group consisting of a mammalian milk di- or oligosaccharide, preferably a human milk di- or oligosaccharide, O-antigen, enterobacterial common antigen (ECA), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG) and antigens of the human ABO blood group system, the method comprising:
• expressing the isolated nucleic acid molecule in a host cell so as to produce the glycosylated product.