US 2023/0416796 A1 · 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.

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(Xn)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(Xn)RXDXD(Xm)[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(Xn)EDD(Xm)[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(Xn)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](Xn)[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.

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Production of galactosylated di- and oligosaccharides

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Production of galactosylated di- and oligosaccharides

Life of this patent

Abstract

Claims58