Microorganisms genetically engineered to have modified N-glycosylation activity
Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.
1. A method of producing an altered N-glycosylation form of a target protein, the method comprising:
providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express an α-1,2-mannosidase polypeptide; and
introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.
2. The method of claim 1 , further comprising isolating the altered N-glycosylation form of the target protein.
3. The method of claim 1 , wherein the target protein is an exogenous protein.
4. The method of claim 1 , wherein the target protein is an endogenous protein.
5. The method of claim 1 , wherein the target protein is a mammalian protein.
6. The method of claim 1 , wherein the target protein is a pathogen protein, a lysosomal protein, a growth factor, a cytokine, a chemokine, or a fusion protein.
7. The method of claim 1 , wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).
8. The method of claim 7 , wherein the lysosomal storage disorder is Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, or Fabry disease.
9. The method of claim 7 , wherein the target protein is selected from the group consisting of glucocerebrosidase, alpha-galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-Dmannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-Nacteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alphaneuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, and lipoprotein lipase.
10. The method of claim 7 , wherein the target protein is human alpha-galactosidase A.
11. The method of claim 1 , wherein the altered N-glycosylation form comprises one or more N-glycan structures selected from group consisting of Man 5 GlcNAc 2 , Man 8 GlcNAc 2 , and Man 9 GlcNAc 2 .
12. The method of claim 1 , wherein the α-1,2-mannosidase polypeptide is a MNS1 polypeptide.
13. The method of claim 1 , wherein the α-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.
14. The method of claim 1 , further comprising additional processing of the target protein in its altered N-glycosylation form.
15. The method of claim 14 , wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.
16. The method of claim 1 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
17. The method of claim 16 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
18. The method of claim 16 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
19. The method of claim 1 , wherein the target protein is a human protein.
20. The method of claim 18 , wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.
21. The method of claim 1 , wherein the α-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R 273 L, R 273 G, R 269 S, and S 272 G.
22. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express an α-1,2-mannosidase polypeptide, wherein the cell is capable of producing a target protein in an altered glycosylation form if a nucleic acid encoding the target protein is introduced into the cell.
23. The cell of claim 22 , said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.
24. The cell of claim 22 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
25. The cell of claim 24 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
26. The cell of claim 24 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
27. The cell of claim 22 , wherein the α-1,2-mannosidase polypeptide is a MNS1 polypeptide.
28. The cell of claim 22 , wherein the α-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.
29. The cell of claim 26 , wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.
30. The cell of claim 22 , wherein the α-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R 273 L, R 273 G, R 269 S, and S 272 G.
31. A method of producing an altered N-glycosylation form of a target protein, the method comprising contacting a target protein with a cell lysate prepared from a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express an α-1,2-mannosidase polypeptide, wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein.
32. The method of claim 31 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
33. The method of claim 32 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
34. The method of claim 32 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
35. The method of claim 31 , wherein the alpha α-1,2-mannosidase polypeptide is a MNS1 polypeptide.
36. The method of claim 31 , wherein the α-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.
37. The method of claim 34 , wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.
38. The method of claim 31 , wherein the α-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R 273 L, R 273 G, R 269 S, and S 272 G.
39. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises a cell genetically engineered to express an α-1,2-mannosidase polypeptide, and wherein the cell is capable of producing a target protein in an altered glycosylation form if a nucleic acid encoding the target protein is introduced into the cell.
40. The substantially pure culture of claim 39 , said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.
41. The substantially pure culture of claim 39 , wherein the α-1,2-mannosidase polypeptide is a MNS1 polypeptide.
42. The substantially pure culture of claim 39 , wherein the α-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.
43. The substantially pure culture of claim 39 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
44. The substantially pure culture of claim 43 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
45. The substantially pure culture of claim 43 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
46. The substantially pure culture of claim 45 , wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.
47. The substantially pure culture of claim 39 , wherein the α-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R 273 L, R 273 G, R 269 S, and S 272 G.