Production of galactosylated di- and oligosaccharides

Abstract

The disclosure is in the technical field of synthetic biology and metabolic engineering. Described is the use of a new type of galactosyltransferases for the production of a galactosylated di- or oligosaccharide. The disclosure also describes methods for the production of a galactosylated di- or oligosaccharide as well as the purification of the di- or oligosaccharide. Furthermore, the disclosure is in the field of cultivation or fermentation of metabolically engineered cells. The disclosure provides a cell metabolically engineered for production of a galactosylated di- or oligosaccharide.

What is claimed is: 
     
         1 . An N-acetylglucosamine b-1,X-galactosyltransferase that galactosylates
 an N-acetylglucosamine and/or N-acetylgalactosamine as a monosaccharide, and/or   an N-acetylglucosamine and/or N-acetylgalactosamine as part of a di- and/or oligosaccharide at the non-reducing end of the di- and/or oligosaccharide, and   
       wherein the N-acetylglucosamine b-1,X-galactosyltransferase is: 
       A. an N-acetylglucosamine b-1,3-galactosyltransferase which has
 a. PFAM domain PF00535 and
 i) comprises the sequence [AGPS]XXLN(X n )RXDXD with SEQ ID NO: 1, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 
 ii) comprises the sequence PXXLN(X n )RXDXD(X m )[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 2, wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an FA, FS, YC or YS combination and wherein n is 12 to 17 and m is 100 to 115, 
 iii) comprises a polypeptide sequence of SEQ ID NO: 3 or 4, or 
 iv) is a functional homologue, variant or derivative of SEQ ID NO: 3 or 4 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, 
 v) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues from SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, 
 vi) is a functional fragment of SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or 
 vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of SEQ ID NO: 3 or 4 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or 
 
 b. PFAM domain IPR002659 and
 i) comprises the sequence KT(Xn)[FY]XXKXDXD(Xm)[FHY]XXG(X, no A, G, S)(Xp)(X, no F, H, W, Y)[DE]D[ILV]XX[AG] with SEQ ID NO: 5, wherein X is any amino acid and wherein n is 13 to 16, m is 35 to 70 and p is 20 to 45, 
 ii) comprises the polypeptide sequence of any one of SEQ ID NOs: 6, 7, 8 or 9, 
 iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 6, 7, 8 or 9 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,3-galactosyltransferase polypeptide with SEQ ID NOs: 6, 7, 8 or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, 
 iv) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, 
 v) is a functional fragment of any one of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or 
 vi) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 6, 7, 8, or 9 and having N-acetylglucosamine b-1,3-galactosyltransferase activity, or 
 
 
       B. an N-acetylglucosamine b-1,4-galactosyltransferase which has
 a. PFAM domain PF01755 and
 i) comprises the sequence EXXCXXSHX[AFILTY]LW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 10, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, or 
 ii) comprises the sequence EXXCXXSH[LR]VLW(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 11, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, 
 iii) comprises the sequence EXXCXXSH[VHI]SLW(X n )EDD(X m )[ACGST]XXY[ILMV] with SEQ ID NO: 12, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, 
 iv) comprises the sequence EXXCXXSHYMLW(X n )EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 13, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, 
 v) comprises the sequence EXXCXXSHXX(X, no V)Y(Xn)EDD(Xm)[ACGST]XXY[ILMV] with SEQ ID NO: 14, wherein X is any amino acid and wherein n is 13 to 15 and m is 50 to 75, 
 vi) comprises the polypeptide sequence of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23, 
 vii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 viii) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 ix) is a functional fragment of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 x) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 15, 18, 22, 20, 17, 19, 16, 21 or 23 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 
 b. PFAM domain PF00535 and
 i) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE] with SEQ ID NO: 24 wherein X is any amino acid and wherein n is 50 to 75 and m is 10 to 30, 
 ii) comprises the sequence R[KN]XXXXXXXGXXXX[FL](X, no V)DXD(Xn)[FHW]XXX[FHNY](Xm)E[DE](Xp)[FWY]XX[HKR]XX[NQST] with SEQ ID NO: 25 wherein X is any amino acid and wherein n is 50 to 75, m is 10 to 30 and p is 20 to 25, 
 iii) comprises a polypeptide sequence of SEQ ID NO: 26 or 27, 
 iv) is a functional homologue, variant or derivative of SEQ ID NO: 26 or 27 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 v) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 vi) is a functional fragment of SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of SEQ ID NO: 26 or 27 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 
 c. PFAM domain PF02709 and not PFAM domain PF00535, and
 i) comprises the sequence [FWY]XX[FWY](X n )[FWY][GQ]X[DE]D with SEQ ID NO: 28 wherein X is any amino acid except for the combination XX on positions 2 and 3 that cannot be an I1P or NL combination and wherein n is 21 to 26, 
 ii) comprises the polypeptide sequence of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34, or 
 iii) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 iv) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 v) is a functional fragment of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 vi) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 33, 29, 30, 31, 32 or 34 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 
 d. PFAM domain PF03808 and
 i) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA with SEQ ID NO: 35, wherein X is any amino acid and wherein n is 20 to 25, 
 ii) comprises the sequence [ST][FHY]XN(Xn)DGXXXXXXXXXXXXXXXX[HKR]X[ST]FDXX[ST]XA (Xm)[HR]XG[FWY](Xp)GXGXXXQ[DE] with SEQ ID NO: 36, wherein X is any amino acid and wherein n is 20 to 25, m is 40 to 50 and p is 22 to 30, 
 iii) comprises the polypeptide sequence of any one of SEQ ID NOs: 37, 38 or 39, 
 iv) is a functional homologue, variant or derivative of any one of SEQ ID NOs: 37, 38 or 39 having at least 80% overall sequence identity to the full length of any one of the N-acetylglucosamine b-1,4-galactosyltransferase polypeptide with SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 v) comprises an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues from any one of SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, 
 vi) functional fragment of any one of SEQ ID NOs: 37, 38 or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity, or 
 vii) comprises a polypeptide comprising a peptide having at least 80% sequence identity to the full-length peptide of any one of SEQ ID NOs: 37, 38, or 39 and having N-acetylglucosamine b-1,4-galactosyltransferase activity. 
 
 
     
     
         2 . A method of synthesizing a galactosylated disaccharide or oligosaccharide, the method comprising:
 utilizing the N-acetylglucosamine b-1,X-galactosyltransferase of  claim 1  in a method of synthesizing a galactosylated disaccharide or oligosaccharide.   
     
     
         3 . The method according to  claim 2 , wherein the synthesis comprises:
 a. providing UDP-galactose and any one of the galactosyltransferase, wherein the galactosyltransferase is capable of transferring a galactose residue from the UDP-galactose donor to one or more acceptor(s), and   b. contacting any one of the galactosyltransferase and UDP-galactose with one or more acceptor(s), under conditions where the galactosyltransferase catalyses the transfer of a galactose residue from the UDP-galactose to the acceptor(s), and   c. optionally, separating the galactosylated di- or oligosaccharide.   
     
     
         4 . The method according to  claim 3 , wherein the acceptor(s) is/are an N-acetylglucosamine and/or an N-acetylgalactosamine as a monosaccharide, and/or a di- and/or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end. 
     
     
         5 . The method according to  claim 2 , wherein the galactosylated disaccharide or oligosaccharide is produced in a cell-free system or is produced by a cell. 
     
     
         6 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell
 is capable of synthesizing one or more of the acceptor(s),   expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases, and   is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases.   
     
     
         7 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of synthesizing one or more nucleotide-sugar donor(s) selected from the group consisting of GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamnine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, UDP-glucuronate, UDP-galacturonate, DP-rhamnose, and UDP-xylose. 
     
     
         8 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of expressing one or more glycosyltransferases selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases. 
     
     
         9 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is a metabolically engineered cell. 
     
     
         10 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is modified in the expression or activity of an enzyme selected from the group consisting of glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase. 
     
     
         11 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate. 
     
     
         12 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is modified for enhanced UDP-galactose production and wherein the modification is selected from the group consisting of knock-out of a 5′-nucleotidase/UDP-sugar hydrolase encoding gene or knock-out of a galactose-1-phosphate uridylyltransferase encoding gene. 
     
     
         13 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell uses at least one precursor for producing the galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium, and/or wherein the cell produces at least one precursor for producing galactosylated disaccharide or oligosaccharide. 
     
     
         14 . The method according to  claim 13 , wherein the precursor for producing galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide. 
     
     
         15 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport. 
     
     
         16 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall. 
     
     
         17 . The method according to  claim 16 , wherein the membrane transporter protein or polypeptide having transport activity:
 controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of at least one precursor and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide, and/or   provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.   
     
     
         18 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor, optionally the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside 0-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase compared to a non-modified progenitor. 
     
     
         19 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of producing phosphoenolpyruvate (PEP), optionally the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor. 
     
     
         20 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for producing galactosylated disaccharide or oligosaccharide. 
     
     
         21 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell resists a phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s). 
     
     
         22 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces 90 g/L or more of the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant and/or wherein the galactosylated disaccharide or oligosaccharide in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of the galactosylated disaccharide or oligosaccharide and its precursor(s) in the whole broth and/or supernatant, respectively. 
     
     
         23 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and wherein the conditions comprise:
 use of a culture medium comprising at least one precursor and/or acceptor for producing galactosylated disaccharide or oligosaccharide, and/or   adding to the culture medium at least one precursor and/or acceptor feed for producing galactosylated disaccharide or oligosaccharide.   
     
     
         24 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell, wherein the culture medium contains at least one precursor selected from the group consisting of lactose, galactose, fucose, and sialic acid. 
     
     
         25 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell, wherein a first phase of exponential cell growth is provided by adding a carbon-based substrate to the culture medium comprising a precursor, followed by a second phase wherein:
 only a carbon-based substrate is added to the culture medium, or   a carbon-based substrate and a precursor are added to the culture medium.   
     
     
         26 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is capable of catabolizing a carbon source selected from the group consisting of glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, corn-steep liquor, molasses, high-fructose syrup, glycerol, acetate, citrate, lactate, and pyruvate. 
     
     
         27 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. 
     
     
         28 . The method according to  claim 27 , wherein the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharides, osmoregulated periplasmic glucans (OPG), glucosylglycerol, glycan, and/or trehalose compared to a non-modified progenitor. 
     
     
         29 . The method according to  claim 2 , wherein the method produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. 
     
     
         30 . The method according to  claim 2 , wherein the method produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide. 
     
     
         31 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. 
     
     
         32 . The method according to  claim 5 , wherein the galactosylated disaccharide or oligosaccharide is produced by a cell and the cell produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide. 
     
     
         33 . The method according to  claim 3 , wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, and ligand exchange chromatography. 
     
     
         34 . The method according to  claim 3 , further comprising purification of the galactosylated di- or oligosaccharide, optionally the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, and vacuum roller drying. 
     
     
         35 . A cell metabolically engineered to synthesize a galactosylated disaccharide or oligosaccharide by utilization of the N-acetylglucosamine b-1,X-galactosyltransferase of  claim 1 . 
     
     
         36 . The cell of  claim 35 , wherein the cell
 expresses any one of the N-acetylglucosamine b-1,3-galactosyltransferases and/or N-acetylglucosamine b-1,4-galactosyltransferases,   is capable of synthesizing UDP-galactose (UDP-Gal) as donor for the galactosyltransferases, and   is capable of synthesizing one or more acceptor(s) for the galactosyltransferases, wherein the acceptor(s) is/are an N-acetylglucosamine as a monosaccharide, and/or a di- or oligosaccharide having an N-acetylglucosamine and/or N-acetylgalactosamine at its non-reducing end.   
     
     
         37 . The cell of  claim 35 , wherein the cell is further capable of synthesizing one or more nucleotide-sugar donor(s) selected from the group consisting of GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-26-dideoxy-L-glucose), GDP-L-quinovose, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N 3 , CMP-Neu4,5AC2, CMP-Neu5,7Ac 2 , CMP-Neu5,9Ac 2 , CMP-Neu5,7(8,9)Ac 2 , UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, and UDP-xylose. 
     
     
         38 . The cell of  claim 35 , wherein the cell is further capable of expressing at least one glycosyltransferase selected from the group consisting of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases. 
     
     
         39 . The cell of  claim 35 , wherein the cell is modified in the expression or activity of an enzyme selected from the group consisting of glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epimerase. 
     
     
         40 . The cell of  claim 35 , wherein the cell is unable to convert N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate, and/or unable to convert glucosamine-6-phosphate to fructose-6-phosphate. 
     
     
         41 . The cell of  claim 35 , wherein the cell is modified for enhanced UDP-galactose production and wherein the modification is selected from the group consisting of knock-out of an 5′-nucleotidase/UDP-sugar hydrolase encoding gene, and knock-out of a galactose-1-phosphate uridylyltransferase encoding gene. 
     
     
         42 . The cell of  claim 35 , wherein the cell uses at least one precursor for producing galactosylated disaccharide or oligosaccharide, the precursor(s) being fed to the cell from the cultivation medium and/or wherein the cell is producing at least one precursor for producing galactosylated disaccharide or oligosaccharide. 
     
     
         43 . The cell of  claim 42 , wherein the precursor for producing galactosylated disaccharide or oligosaccharide is completely converted into the galactosylated disaccharide or oligosaccharide. 
     
     
         44 . The cell of  claim 35 , wherein the cell produces the galactosylated disaccharide or oligosaccharide intracellularly and wherein a fraction or substantially all of the produced galactosylated disaccharide or oligosaccharide remains intracellularly and/or is excreted outside the cell via passive or active transport. 
     
     
         45 . The cell of  claim 35 , wherein the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall. 
     
     
         46 . The cell of  claim 45 , wherein the membrane transporter protein or polypeptide having transport activity:
 controls the flow over the outer membrane of the cell wall of the galactosylated disaccharide or oligosaccharide and/or of at least one precursor and/or acceptor(s) to be used in the production of the galactosylated disaccharide or oligosaccharide, and/or   provides improved production and/or enabled and/or enhanced efflux of the galactosylated disaccharide or oligosaccharide.   
     
     
         47 . The cell of  claim 35 , wherein the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor, optionally the cell comprises a lower or reduced expression and/or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside 0-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, lon protease, glucose-specific translocating phosphotransferase enzyme IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, and pyruvate decarboxylase compared to a non-modified progenitor. 
     
     
         48 . The cell of  claim 35 , wherein the cell is capable of producing phosphoenolpyruvate (PEP), optionally the cell is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor. 
     
     
         49 . The cell of  claim 35 , wherein the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for producing galactosylated disaccharide or oligosaccharide. 
     
     
         50 . The cell of  claim 35 , wherein the cell resists the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s). 
     
     
         51 . The cell of  claim 35 , wherein the cell is capable of catabolizing a carbon source selected from the group consisting of glucose, fructose, mannose, galactose, lactose, sucrose, maltose, malto-oligosaccharides, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, glycerol, acetate, citrate, lactate, and pyruvate. 
     
     
         52 . The cell of  claim 35 , wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. 
     
     
         53 . The cell of  claim 52 , wherein the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharides, osmoregulated periplasmic glucans (OPG), glucosylglycerol, glycan, and/or trehalose compared to a non-modified progenitor. 
     
     
         54 . The cell of  claim 35 , wherein the cell produces a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide. 
     
     
         55 . The cell of  claim 35 , wherein the cell produces a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide. 
     
     
         56 . A method of producing a galactosylated disaccharide or oligosaccharide, the method comprising:
 cultivating the cell of  claim 35  so as to produce a galactosylated disaccharide or oligosaccharide.   
     
     
         57 . A method of producing a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide, the method comprising:
 cultivating the cell of  claim 35  so as to produce a mixture of charged and/or neutral di- and/or oligosaccharides comprising at least one galactosylated disaccharide or oligosaccharide.   
     
     
         58 . A method of producing a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide, the method comprising:
 cultivating the cell of  claim 35  so as to produce a mixture of charged and/or neutral oligosaccharides comprising at least one galactosylated oligosaccharide.