Fucosyltransferases and their applications
The present invention relates to nucleic acid and amino acid sequences from Akkermansia muciniphila and from Bacteroides fragilis , coding for/representing novel alpha-1,3-fucosyltransferases. The invention also provides uses and methods for using the alpha-1,3-fucosyltransferases to generate fucosylated products, such as oligosaccharides, (glyco)proteins, or (glyco)lipids, in particular of 3-fucosyllactose.
1. A method for producing fucosylated oligosaccharides, the method comprising the steps of:
a. providing a polypeptide with alpha-1,3-fucosyltransferase activity and with the ability to use lactose, or an oligosaccharide comprising a lactose residue, as acceptor substrate,
wherein the polypeptide consists of the amino acid sequence set forth as one of SEQ ID NO. 2, SEQ ID NO: 4, SEQ ID NO: 6, an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO. 2, an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 4, or an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 6; and
b. contacting the polypeptide with alpha-1,3-fucosyltransferase activity of step a. with a mixture comprising a donor substrate comprising a fucose residue, and an acceptor substrate comprising lactose or an oligosaccharide comprising a lactose residue, under conditions where the polypeptide catalyzes the transfer of a fucose residue from the donor substrate to the acceptor substrate,
thereby producing fucosylated oligosaccharides.
2. A method for producing fucosylated oligosaccharides, comprising the steps of:
a. growing in vitro, a bacterial or fungal host cell comprising a vector, wherein the vector comprises a nucleic acid sequence encoding a polypeptide with alpha-1,3-fucosyltransferase activity and with the ability to use lactose, or an oligosaccharide comprising a lactose residue as an acceptor substrate, the nucleic acid sequence being operably linked to a control sequence, wherein the nucleic acid sequence is selected from the group consisting of the nucleic acid sequence set forth as SEQ ID NO: 1, the nucleic acid sequence set forth as SEQ ID NO: 3 the nucleic acid sequence set forth as SEQ ID NO: 5, a nucleic acid sequence complementary to the nucleic acid sequence set forth as SEQ ID NO: 1, a nucleic acid sequence complementary to the nucleic acid sequence set forth as SEQ ID NO: 3, and a nucleic acid sequence complementary to the nucleic acid sequence set forth as SEQ ID NO: 5;
b. providing simultaneously or subsequently to step a, a donor substrate comprising a fucose residue and an acceptor substrate comprising lactose or an oligosaccharide comprising a lactose residue, in order for the alpha-1,3-fucosyltransferase polypeptide to catalyze the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing a fucosylated oligosaccharide, and
c. isolating said fucosylated oligosaccharide from the host cell or a medium of its growth.
3. The method of claim 1 , wherein the donor substrate is GDP-fucose.
4. The method of claim 2 , wherein the donor substrate is GDP-fucose.
5. The method of claim 3 , wherein the GDP-fucose is provided by an enzyme simultaneously expressed in the host cell or by the metabolism of the host cell.
6. The method of claim 4 , wherein the GDP-fucose is provided by an enzyme simultaneously expressed in the host cell or by the metabolism of the host cell.
7. The fucosylated oligosaccharide obtained by the method of claim 1 .
8. The fucosylated oligosaccharide obtained by the method of claim 2 .
9. A method for producing a fucosyllactose using a bacterial or fungal host cell, comprising the steps of:
a. growing, in vitro, a bacterial or fungal host cell transformed or transfected to express an exogenous polypeptide with alpha-1,3-fucosyltransferase activity and with the ability to use lactose as an acceptor substrate, wherein the polypeptide comprises the amino acid sequence set forth as SEQ ID NO. 2, the amino acid sequence set forth as SEQ ID NO: 4, the amino acid sequence set forth as SEQ ID NO: 6, an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO. 2, an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 4, or an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 6; and
i) an exogenous polypeptide with a bifunctional fucose-1-phosphate guanylyltransferase/fucose kinase activity or ii) both an exogenous polypeptide with fucose-l-phosphate guanylyltransferase activity and an exogenous polypeptide with fucose kinase activity;
b. providing, simultaneously or subsequently to step a, a donor substrate comprising a fucose residue and an acceptor substrate, wherein the acceptor substrate is selected from a mono- or disaccharide or a combination thereof, wherein the alpha-1,3-fucosyltransferase polypeptide catalyzes the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing a fucosyllactose; and
c. isolating the fucosyllactose from the host cell or a medium of its growth.
10. The method of claim 1 , wherein the produced fucoslyated oligosaccharide is 3-fucosyllactose.
11. The method of claim 2 , wherein the produced fucosylated oligosaccharide is 3-fucosyllactose.
12. The method of claim 9 , wherein the exogenous polypeptide comprises the amino acid sequence set forth as SEQ ID NO. 2, SEQ ID NO: 4, or SEQ ID NO: 6.
13. The method of claim 9 , wherein the bacterial or fungal host cell is transformed with a nucleic acid molecule encoding the exogenous polypeptide, and wherein the nucleic acid molecule comprises the nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
14. The method of claim 2 , wherein the host cell is an E. coli cell or a fungal cell.
15. The method of claim 5 , wherein the host cell is an E. coli cell or a fungal cell.
16. The method of claim 9 , wherein the produced fucosyllactose is 3-fucosyllactose.
17. The method of claim 9 , wherein the polypeptide consists of the amino acid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
18. The method of claim 9 , wherein the polypeptide consists of the amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO. 2, the amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 4, or the amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 6.