Methods for producing modified glycoproteins

A yeast or filamentous fungal host cell that does not have alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the host cell comprising one or more nucleic acids encoding at least two enzymes, the first enzyme selected to have optimal activity in the endoplasmatic reticulum (ER) or Golgi of said host cell, the first enzyme comprising:

• (a) an alpha-1,2 mannosidase catalytic domain having optimal alpha-1,2 mannosidase activity in said ER or Golgi at a pH between 5.1 and 8.0;
• fused to
• (b) a cellular targeting signal peptide not normally associated with the catalytic domain of (a) and selected to target the first enzyme to the ER or Golgi apparatus of the host cell;
• the second enzyme selected to have optimal activity in the ER or Golgi of said host cell, the second enzyme comprising:
• (c) a GlcNAc transferase I catalytic domain having optimal GIcNAc transferase I activity in said ER or Golgi at a pH between 5.1 and 8.0;
• fused to
• (d) a cellular targeting signal peptide not normally associated with the catalytic domain of (c) and selected to target the second enzyme to the ER or Golgi apparatus of the host cell,
• wherein said host cell is capable of producing recombinant glycoproteins comprising a GlcNAcMan5GlcNAc2 structure.

A yeast host cell that does not have an alpha-1,6 mannosyltransferase activity with respect to an N-glycan on a glycoprotein, comprising a nucleic acid encoding an alpha-1,2 mannosidase enzyme selected to have optimal activity in the endoplasmic reticulum (ER) or Golgi of said host cell, the enzyme comprising:

• (a) an alpha-1,2 mannosidase catalytic domain having optimal alpha-1,2 mannosidase activity in said ER or Golgi at a pH between 5.1 and 8.0;
• fused to
• (b) a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the mannosidase catalytic domain to said ER or Golgi apparatus;
• wherein said host cell is capable of producing recombinant glycoproteins wherein in excess of 30 mole % of the N-glycan structures attached thereto have a Man5GlcNAc2 structure that can serve as a substrate for GlcNAc transferase I in vivo.

A yeast host cell that does not have alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the host cell comprising one or more nucleic acids encoding at least two enzymes, the first enzyme selected to have optimal activity in the endoplasmatic reticulum (ER) or Golgi of said host cell, the first enzyme comprising:

• (a) an alpha-1,2 mannosidase catalytic domain having optimal alpha-1,2 mannosidase activity in said ER or Golgi at a pH between 5.1 and 8.0;
• fused to
• (b) a cellular targeting signal peptide not normally associated with the catalytic domain of (a) and selected to target the first enzyme to the ER or Golgi apparatus of the host cell;
• the second enzyme selected to have optimal activity in the ER or Golgi of said host cell, the second enzyme comprising:
• (c) a GlcNAc transferase I catalytic domain having optimal activity GlcNAc transferase I activity in said ER or Golgi at a pH between 5.1 and 8.0;
• fused to
• (d) a cellular targeting signal peptide not normally associated with the catalytic domain of (c) and selected to target the second enzyme to the ER or Golgi apparatus of the host cell, wherein said host cell is capable of producing recombinant glycoproteins comprising a GlcNAcMan5GlcNAc2 structure.