Sialyltransferases and their use in producing sialylated oligosaccharides
Disclosed are methods, genetically engineered cells, sialyltransferases and nucleic acid molecules encoding said sialyltransferases for producing sialylated oligosaccharides as well as the use of said sialylated oligosaccharides for providing nutritional compositions.
1 . A method for producing one or more sialylated oligosaccharides by whole cell fermentation, the method comprising:
a) providing at least one genetically engineered cell which comprises a heterologous sialyltransferase, wherein said heterologous sialyltransferase possesses an α-2,3-sialyltransferase activity and/or an α-2,6-sialyltransferase activity for transferring a sialic acid residue from a nucleotide-activated form as donor substrate to an acceptor molecule,
wherein the acceptor molecule is lacto-N-tetraose, and
wherein the heterologous sialyltransferase is selected from
i) polypeptides comprising an amino acid sequence of any one of SEQ ID NOs: 34, 36, 62 and 63; and
ii) fragments of polypeptides comprising an amino acid sequence of any one of SEQ ID NOs: 34, 36, 62 and 63; and
b) cultivating the at least one cell in a fermentation broth and under conditions permissive for the production of said sialylated oligosaccharide during cultivation in the fermentation broth; and
c) recovering said sialylated oligosaccharide from the fermentation broth.
2 . The method according to claim 1 , wherein the sialylated oligosaccharide is selected from sialyllacto-N-tetraose a (LST-a), sialyllacto-Ntetraose b (LST-b), sialyllacto-N-tetraose c (LST-c), and disialyllacto-N-tetraose (DSLNT).
3 . The method according to claim 1 , wherein the fermentation broth comprises at least one carbon source.
4 . The method according to claim 1 , wherein the fermentation broth comprises at least one of N-acetylglucosamine, galactose and sialic acid.
5 . The method according to claim 1 , wherein the at least one genetically engineered cell is cultivated in the absence of and/or without addition of one or more of N-acetylglucosamine, galactose and sialic acid.
6 . The method according to claim 1 , wherein the at least one cell is cultivated in the presence of at least one of lactose, lacto-N-triose II, LNT and LNnt.
7 . The method according to claim 1 , wherein the genetically engineered cell comprises a nucleic acid molecule comprising a nucleotide sequence which encodes the heterologous sialyltransferase, said nucleotide sequence being selected from:
i) nucleotide sequences as represented by any one of SEQ ID NOs: 1, 3, 29 and 30;
ii) nucleotide sequences encoding a polypeptide as represented by any one of SEQ ID NOs: 34, 36, 62 and 63;
iii) nucleotide sequences encoding a fragment of a polypeptide as represented by any one of SEQ ID NOs: 34, 36, 62 and 63;
iv) nucleotide sequences which are complementary to any one of the nucleotide sequences of i, ii, and iii,
wherein said nucleotide sequence is operably linked to at least one nucleic acid expression control sequence effecting transcription and/or translation of said nucleotide sequence encoding heterologous sialyltransferase in the genetically engineered cell.
8 . The method according to claim 1 , wherein the genetically engineered cell possesses an increased production of one or more nucleotideactivated sugars selected from CMP-N-acetylneuraminic acid, UDP-Nacetylglucosamine, UDP-galactose and GDP-fucose as compared to the cell before being genetically engineered.
9 . The method according to claim 1 , wherein the genetically engineered cell possesses an overexpression, as compared to the cell prior to being genetically engineered, of one or more genes encoding for a polypeptide that possesses an enzymatic activity selected from L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, glucosamine-6-phosphate-N-acetyltransferase, N-acetylglucosamine-2-epimerase, UDP-Nacetylglucosamine-2-epimerase, sialic acid synthase, phosphoenolpyruvate synthase, CMP-sialic acid synthetase, UDP-galactose-4-epimerase, galactose-1-phosphate uridylyltransferase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase, phosphomannomutase, mannose-1-phosphate guanosyltransferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase and fucosekinase/L-fucose-1-phosphateguanyltransferase.
10 . The method according to claim 1 , wherein the cell lacks or possesses a decreased activity, as compared to the cell prior to being genetically engineered, of one or more enzymatic activities selected from β-galactosidase, a glucosamine-6-phosphate deaminase, a N-acetylglucosamine-6-phosphate deacetylase, a N-acetylmannosamine kinase, a N-acetylmannosamine-6-phosphate epimerase and a N-acetylneuraminic acid aldolase.
11 . The method according to claim 1 , wherein one or more of the genes encoding a β-galactosidase, a glucosamine-6-phosphate deaminase, a N-acetylglucosamine-6-phosphate deacetylase, a N-acetylmannosamine kinase, a N-acetylmannosamine-6-phosphate epimerase and a N-acetylneuraminic acid aldolase has been deleted or expression thereof has been inactivated in the genetically engineered cell.
12 . The method according to claim 1 , wherein the at least one cell comprises at least one of a functional lactose permease, a functional fucose permease and a functional sialic acid transporter.
13 . The method according to claim 1 , wherein the cell possesses activity of at least one glycosyltransferase selected from a β-1,3-N acetylglucosaminyltransferase, a β-1,3-galactosyltransferase, a β-1,4-galactosyltransferase, a α-2,3-sialyltransferase and a α-2,6-sialyltransferase.
14 . The method according to claim 3 , wherein the at least one carbon source is glucose, fructose, sucrose, glycerol, or combinations thereof.
15 . The method according to claim 12 , wherein the at least one cell further comprises and expresses at least one nucleotide sequence encoding one of a functional lactose permease, a functional fucose permease, and a functional sialic acid transporter.
16 . The method of claim 1 , wherein the heterologous sialyltransferase has a relative efficacy of at least 100-fold compared to the efficacy of a sialyltransferase reference sequence, which is an α-2,3-sialyltransferase of SEQ ID NO: 60 or an α-2,6-sialyltransferease of SEQ ID NO: 66.