Method to engineer mammalian-type carbohydrate structures
The present invention relates to host cells having modified lipid-linked oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells have a GlcNAcMan 3 GlcNAc 2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.
1. A method for producing a recombinant glycoprotein in a Pichia pastoris host cell that lacks OCH1 activity, the method comprising the steps of diminishing or depleting the activity of one or more enzymes-selected from the group consisting of:
(a) an enzyme having dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity;
(b) an enzyme having dolichyl-P-Man:Man 6 GlcNAc 2 -PP-dolichyl alpha-1,2 mannosyltransferase activity; and
(c) an enzyme having dolichyl-P-Man:Man 7 GlcNAc 2 -PP-dolichyl alpha-1,6 mannosyltransferase activity,
said host cell further expressing: (i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and (iii) the recombinant glycoprotein,
wherein said method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAcMan x GlcNAc 2 core structures, wherein X is 3 or 4.
2. The method of claim 1 , wherein the host cell further includes a nucleic acid molecule encoding a mannosidase II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAcMan 3 GlcNAc 2 core structures.
3. The method of claim 2 , wherein the host cell further includes a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc 2 Man 3 GlcNAc 2 core structures.
4. The method of claim 1 wherein the host cell further includes a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc 2 Man 3 GlcNAc 2 core structures.
5. The method of claim 4 , wherein the wherein the host cell further includes one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI.
6. The method of claim 1 , wherein the diminished or depleted enzyme has dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity.
7. The method of claim 1 , wherein the enzyme is diminished or depleted by mutation of a host cell gene encoding the enzymatic activity.
8. The method of claim 7 , wherein the mutation is a partial or total deletion of a host cell gene encoding the enzymatic activity.
9. The method of claim 1 , wherein the attached N glycans have seven or fewer mannose residues.
10. The method of claim 1 , wherein the glycoprotein comprises one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose.
11. The method of claim 1 , wherein the glycoprotein comprises at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man.
12. A method for producing a human-like glycoprotein in a Pichia pastoris host cell that lacks OCH1 activity comprising the steps of diminishing or depleting from the host cell an alg gene activity selected from the group consisting of:
(a) an enzyme having dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity,
(b) an enzyme having dolichyl-P-Man:Man 6 GlcNAc 2 -PP-dolichyl alpha-1,2 mannosyltransferase activity, and
(c) an enzyme having dolichyl-P-Man:Man 7 GlcNAc 2 -PP-dolichyl alpha-1,6 mannosyltransferase activity;
and introducing into the host cell at least one glycosidase activity.
13. A method for producing a recombinant glycoprotein in a Pichia pastoris host cell, the method comprising:
(a) providing a Pichia pastoris host cell that lacks OCH1 activity and that has diminished or depleted activity of one or more enzymes selected from the group consisting of:
(a) an enzyme having dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity,
(b) an enzyme having dolichyl-P-Man:Man 6 GlcNAc 2 -PP-dolichyl alpha-1,2 mannosyltransferase activity, and
(c) an enzyme having dolichyl-P-Man:Man 7 GlcNAc 2 -PP-dolichyl alpha-1,6 mannosyltransferase activity,
said host cell further expressing:
(i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell,
(ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and
(iii) a recombinant glycoprotein; and
(b) growing the host cell under conditions to produce the recombinant glycoprotein, wherein the recombinant glycoprotein has N-glycans attached thereto comprising GlcNAcMan x GlcNAc 2 core structures, wherein X is 3 or 4.
14. The method of claim 13 , wherein the host cell further includes a nucleic acid molecule encoding a mannosidase II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and wherein the recombinant glycoprotein that is produced has N-glycans attached thereto comprising GlcNAcMan 3 GlcNAc 2 core structures.
15. The method of claim 14 , wherein the host cell further includes a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and wherein the recombinant glycoprotein that is produced has N-glycans attached thereto comprising a GlcNAc 2 Man 3 GlcNAc 2 structure.
16. The method of claim 13 , wherein the host cell further includes a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and wherein the recombinant glycoprotein that is produced has N-glycans attached thereto comprising a GlcNAc 2 Man 3 GlcNAc 2 structure.
17. The method of claim 13 , wherein the host cell further includes one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.
18. The method of claim 13 , wherein the host cell further includes one or more nucleic acid molecules encoding at least one enzyme activity selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI.
19. The method of claim 13 , wherein the diminished or depleted enzyme has dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity.
20. The method of claim 13 , wherein the diminished or depleted enzyme activity is by mutation of a host cell gene encoding the enzymatic activity.
21. The method of claim 20 , wherein the mutation is a partial or total deletion of a host cell gene encoding the enzymatic activity.
22. The method of claim 13 , wherein the glycoprotein comprises one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose.
23. The method of claim 13 , wherein the glycoprotein comprises at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man.
24. A method for producing a recombinant glycoprotein in a Pichia pastoris host cell, the method comprising
(a) providing a Pichia pastoris host cell in which OCH1 activity and dolichyl-P-Man:Man 5 GlcNAc 2 -PP-dolichyl alpha-1,3 mannosyltransferase activity of the host cell have been diminished or depleted, and which comprises one or more nucleic acid molecules encoding
(i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell,
(ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and
(iii) a recombinant glycoprotein; and
(b) growing the host cell under conditions to produce the recombinant glycoprotein, wherein the recombinant glycoprotein has N-glycans attached thereto comprising GlcNAcMan 3 GlcNAc 2 core structures.
25. The method of claim 24 , wherein the host cell further includes a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and wherein the recombinant glycoprotein that is produced has N-glycans attached thereto comprising a GlcNAc 2 Man 3 GlcNAc 2 structure.
26. The method of claim 24 , wherein the host cell further includes one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNac transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.
27. The method of claim 24 , wherein the host cell further includes one or more nucleic acid molecules encoding at least one enzyme activity selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, andr VI.
28. The method of claim 24 , wherein the diminished or depleted enzyme activity is by mutation of a host cell gene encoding the enzymatic activity.
29. The method of claim 8 , wherein the mutation is a partial or total deletion of a host cell gene encoding the enzymatic activity.
30. The method of claim 24 , wherein the glycoprotein comprises one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose.
31. The method of claim 24 , wherein the glycoprotein comprises at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man.