Methods of glycoengineering proteoglycans with distinct glycan structures

A method of generating a glycoprotein comprising a distinct glycan structure, said method comprising expressing at least one protein of interest and at least one glycan modifying enzyme in an engineered, non-naturally occurring eukaryotic cell comprising a modified genome, wherein the modified genome comprises:

• (a) a knockout of at least one endogenous polynucleic acid sequence encoding a glycan modifying enzyme;
• (b) an integration of at least one polynucleic acid sequence comprising a sequence encoding a functional copy of a glycan modifying enzyme knocked out in (a), wherein the sequence encoding the functional copy of a glycan modifying enzyme is operably linked to a tunable control element that controls mRNA and/or protein expression of the glycan modifying enzyme;
• and
• (c) an integration of at least one polynucleic acid sequence comprising a sequence encoding a protein of interest operably linked to a constitutive or inducible promoter, wherein each of the at least one protein of interest can, when expressed as a protein, be modified by the addition of a glycan.