Fucosyltransferases and their applications
The present invention relates to nucleic acid and amino acid sequences from Escherichia coli serogroup O126, coding for/representing a novel alpha-1,2-fucosyltransferase. The invention also provides uses and methods for using the alpha-1,2-fucosyltransferase to generate fucosylated products, such as oligosaccharides, (glyco)proteins, or (glyco)lipids, in particular oligosaccharides found in human milk, such as 2′-fucosyllactose.
1. A method for producing fucosyllactose comprising the steps of:
a) providing a polypeptide with alpha-1,2-fucosyltransferase activity comprising the amino acid sequence shown in SEQ ID NO: 2, wherein the polypeptide can transfer a fucose residue to a monosaccharide or an oligosaccharide that comprises lactose; and
b) contacting the polypeptide with alpha-1,2-fucosyltransferase activity of step a) with a mixture comprising a donor substrate, wherein the donor substrate is GDP-fucose, and an acceptor substrate comprising a mono- or oligosaccharide comprising lactose under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing the fucosyllactose.
2. A method for producing fucosyllactose comprising the steps of:
a) growing, under suitable nutrient conditions permissive for the production of the fucosylated oligosaccharide, and permissive for the expression of a polypeptide with alpha-1,2-fucosyltransferase activity, a host cell, wherein the host cell comprises a polynucleotide encoding a polypeptide with alpha-1,2-fucosyltransferase activity wherein the polynucleotide is selected from the group consisting of (i) the nucleic acid sequence shown in SEQ ID NO: 1, or (ii) a nucleic acid sequence complementary to SEQ ID NO: 1, wherein the polynucleotide is foreign to the host cell and wherein the polynucleotide is integrated in the genome of the host cell or wherein the host cell comprises a vector comprising the polynucleotide operably linked to control sequences recognized by the host cell transformed with the vector;
b) providing, simultaneously or subsequently to step a), a donor substrate wherein the donor substrate is a GDP-fucose residue, and an acceptor substrate comprising a mono- or oligosaccharide comprising lactose, in order for the alpha-1,2-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing fucosyllactose; and
c) isolating said fucosyllactose from the host cell or the medium of its growth.
3. The method according to claim 2 , wherein the GDP-fucose residue is provided by an enzyme simultaneously expressed in the host cell or by the metabolism of the host cell.
4. The method of claim 2 , wherein the fucosyllactose is 2′-fucosyllactose.
5. A method for producing a fucosyllactose comprising expressing a polynucleotide encoding a polypeptide with alpha-1,2-fucosyltransferase activity, comprising:
contacting a host cell comprising a polynucleotide encoding a polypeptide with alpha-1,2-fucosyltransferase activity with a donor substrate,
wherein the polynucleotide is selected from the group consisting of a) the nucleic acid sequence shown in SEQ ID NO: 1, or b) a nucleic acid sequence complementary to SEQ ID NO: 1,
wherein the polynucleotide is foreign to the host cell and wherein the polynucleotide is integrated in the genome of the host cell or wherein the host cell comprises a vector comprising the polynucleotide operably linked to control sequences recognized by the host cell transformed with the vector, and
wherein the donor substrate comprises a GDP-fucose residue or is GDP-fucose.
6. The method according to claim 5 , wherein the GDP-fucose residue is provided by an enzyme simultaneously expressed in the host cell or by the metabolism of the host cell.
7. The method of claim 5 , wherein the fucosyllactose is 2′-fucosyllactose.
8. The method of claim 2 , wherein the host cell is selected from the group consisting of fungal, yeast, bacteria, insect, animal and plant cells.
9. The method of claim 2 , wherein the host cell is an Escherichia coli cell.
10. The method of claim 2 , wherein the nucleic acid sequence encoding the polypeptide with alpha-1,2-fucosyltransferase activity is adapted to the codon usage of the cell.
11. The method of claim 2 , wherein the polynucleotide encoding a polypeptide with alpha-1,2-fucosyltransferase activity comprises the nucleic acid sequence set forth as SEQ ID NO: 1.
12. A method for producing fucosyllactose comprising the steps of:
a) growing, under suitable nutrient conditions permissive for the production of the fucosylated oligosaccharide, permissive for the expression of a polypeptide with alpha-1,2-fucosyltransferase activity, a host cell with the ability to transfer a fucose residue to a mono- or oligosaccharide comprising lactose, wherein the host cell expresses a polypeptide with alpha-1,2-fucosyltransferase activity and the ability to transfer a fucose residue to a mono- or oligosaccharide comprising lactose, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2;
b) providing, simultaneously or subsequently to step a), a donor substrate wherein the donor substrate is a GDP-fucose residue, and an acceptor substrate comprising a mono- or oligosaccharide comprising lactose, in order for the alpha-1,2-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing fucosyllactose; and
c) isolating said fucosyllactose from the host cell or the medium of its growth.
13. The method of claim 12 , wherein the polypeptide with alpha-1,2-fucosyltransferase activity consists of the amino acid sequence shown in SEQ ID NO: 2.
14. The method of claim 1 , wherein the polypeptide with alpha-1,2-fucosyltransferase activity consists of the amino acid sequence shown in SEQ ID NO: 2.
15. The method of claim 5 , wherein the host cell is an Escherichia coli cell.
16. The method of claim 12 , wherein the host cell is an Escherichia coli cell.