Production of human milk oligosaccharides in microbial hosts with engineered import / export
The present invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as to the genetically modified host cells used in the methods. The genetically modified host cell comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling the export of the oligosaccharide.
1. A method for production of an oligosaccharide by a genetically modified microbial host cell, wherein the oligosaccharide is a lacto-N-tetraose comprising a lacto-N-triose II (LNT-II; GlcNAc(b1-3)Gal(b1-4)Gluc) as a core trisaccharide, the method comprising:
providing a genetically modified microbial host cell that comprises:
at least one recombinant glycosyltransferase selected from the group consisting of a β-1,3-N-acetylglucosaminyltransferase and a β-1,3-galactosyltransferase;
increased expression or activity of at least one homologous or heterologous sugar export protein capable of exporting the oligosaccharide; and
decreased expression or inactivation of at least one export protein that exports LNT-II from the host cell;
cultivating the host cell in a medium under conditions permissive for the production of the oligosaccharide, whereby the oligosaccharide is exported into the medium at an increased level compared to an unmodified host cell, and
obtaining the oligosaccharide from the medium.
2. The method of claim 1 , wherein the host cell further comprises:
overexpression of at least one homologous or heterologous nucleic acid sequence coding for the sugar export protein capable of exporting the oligosaccharide into the culture medium; and/or
deletion, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an export protein that exports precursors of the oligosaccharide outside the host cell; and/or
overexpression of at least one homologous or heterologous sequence coding for a protein mediating import of a precursor of the oligosaccharide into said host cell, wherein the precursor is larger than a disaccharide.
3. The method of claim 2 , wherein said sugar export protein capable of exporting the oligosaccharide belongs to the class of secondary active transporters.
4. The method of claim 2 , wherein the nucleic acid sequence coding for the homologous or heterologous sugar export protein capable of exporting the oligosaccharide is of bacterial, archeal, plant, yeast or animal origin.
5. The method of claim 2 , wherein said at least one nucleic acid sequence coding for the sugar export protein that exports precursors of the oligosaccharide outside the host cell is a gene selected from the group consisting of yebQ from Escherichia coli , yjhB from Escherichia coli , proP from Mannheimia succiniciproducens , and setA from Cedecea neteri.
6. The method of claim 1 , wherein said β-1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningitides or PmnagT of Pasteurella multocida.
7. The method of claim 1 , wherein the β-1,3-galactosyltransferase is encoded by a WbdO gene.
8. The method of claim 1 , wherein-ii the genetically modified microbial host cell further comprises an endogenous β-galactosidase gene and glucosamine-6-phosphate deaminase gene that are inactivated or deleted, and wherein said genetically modified microbial host cell further comprises a nucleic acid sequence coding for a functional lactose permease protein.
9. The method of claim 1 , wherein the genetically modified microbial host cell further comprises an increased UDP-N-acetylglucosamine and a UDP-galactose, or GDP-fucose, or CMP-N-acetylneuraminic acid production, as compared to a genetically unmodified host cell, wherein optionally said increased UDP-N-acetylglucosamine and UDP-galactose production capability is by an overexpression of one or more genes encoding a protein selected from the group consisting of L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, and glucose-1-phosphate uridylyltransferase.
10. The method of claim 8 , wherein said genetically modified microbial host cell is cultivated in the presence of glucose, sucrose, glycerol or a combination thereof.
11. The method of claim 10 , wherein the microbial host cell is cultured in the absence of N-acetylglucosamine and galactose.
12. A genetically modified microbial host cell for the production of lacto-N-tetraose comprising a lacto-N-triose II (LNT-II; GlcNAc(β1-3)Gal(β1-4)Gluc) as a core trisaccharide, wherein the host cell comprises
at least one recombinant glycosyltransferase, selected from the group consisting of a β-1,3-N-acetylglucosaminyltransferase and a β-1,3-galactosyltransferase;
at least one homologous or heterologous nucleic acid sequence coding for a sugar export protein capable exporting of the acto-N-tetraose into a culture medium the host cell is cultivated in wherein said sugar export protein capable of exporting the lacto-N-tetraose belongs to the class of secondary active transporters, and wherein said homologous or heterologous nucleic acid sequence coding for the sugar export protein capable of exporting the lacto-N-tetraose is overexpressed or under control of a promoter sequence enabling the overexpression of said nucleic acid sequence; and
inactivated expression or activity of at least one endogenous sugar export protein that exports LNT-II from the host cell.
13. The microbial host cell of claim 12 , further comprising an overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein mediating the import of a precursor of the acto-N-tetraose into said host cell through genetic modification of the microbial host cell, wherein the precursor is larger than a disaccharide.
14. The microbial host cell of claim 12 , wherein said nucleic acid sequence coding for the sugar export protein capable of exporting the acto-N-tetraose is of bacterial, archeal, plant, yeast or animal origin.
15. The microbial host cell of claim 12 , wherein said at least one nucleic acid sequence coding for the export protein that exports LNT-II from the host cell is a gene selected from the group consisting of yebQ from Escherichia coli , yjhB from Escherichia coli , proP from Mannheimia succiniciproducens , and setA from Cedecea neteri.
16. The microbial host cell of claim 14 , wherein the sugar export protein capable of exporting the lacto-N-tetraose is selected from the group consisting of YebQ from Escherichia coli BL21(DE3), SpoVB of Bacillus amyloliquefaciens , YabM of Erwinia pyrilfolia , Bcr of E. coli MG1655, YdeA of E. coli MG1655, ProP2 of Haemophilus parainfluenzae , SetA of Pectobacterium carotovorum , FucP of E. coli MG1655, MdeA of Staphylococcus aureus Bmb9393, ImrA of Lactococcus lactis , SetA of Pseudomonas sp. MT-1 and SetA of Beauveria bassiana Dl-5.
17. The method of claim 1 , wherein the sugar export protein capable of exporting the lacto-N-tetraose is selected from the group consisting of YebQ from Escherichia coli BL21(DE3), SpoVB of Bacillus amyloliquefaciens , YabM of Erwinia pyrilfolia , Bcr of E. coli MG1655, YdeA of E. coli MG1655, ProP2 of Haemophilus parainfluenzae , SetA of Pectobacterium carotovorum , FucP of E. coli MG1655, MdeA of Staphylococcus aureus Bmb9393, ImrA of Lactococcus lactis , SetA of Pseudomonas sp. MT-1 and SetA of Beauveria bassiana D1-5.
18. The method of claim 1 , wherein the microbial host cell is an Escherichia coli cell, a Corynebacterium glutamicum cell or a Saccharomyces sp. cell.
19. The microbial host cell of claim 12 , wherein the microbial host cell is an Escherichia coli cell, a Corynebacterium glutamicum cell or a Saccharomyces sp. cell.