Table 5.

Genes that have been engineered to improve glycan pattern homogeneity and glycosylation efficiency in the HEK293 cell line.

Gene	Function	Modification	Outcome	Recombinant Protein(s)	Reference
N-acetylglucosaminyltransferase I (GnTI) protein encoded by the MGAT1 gene	Processing of high-mannose to hybrid and complex N-glycans	Knockout	Inability to synthesize complex glycans. Only the Man5GlcNAc2 N-Glycan present	Rhodopsin	[87]
Endo-β-N-acetylglucosaminidase from Hypocrea jecorina (endoT8) fused to the Golgi targeting domain of the human β-galactoside-α-2,6-sialyltransferase 1 (in GnTI-/- cells)	Catalyzes the hydrolysis of mannose modifications to produce free oligosaccharides. Catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates	Overexpression	Improved homogeneity in glycan expression	Granulocyte macrophage colony-stimulating factor (GM-CSF), monoclonal anti-CD20 antibody GA101	[90]
Golgi mannosidases MAN1A1	Catalyzes the removal of 3 distinct mannose residues from peptide-bound Man (9)-GlcNAc (2) oligosaccharides.	Triple knockout	Production of simple Man9GlcNAc2 and Man8GlcNAc2 structures of high-Man–type glycans; limited complex-type N-glycans at relatively low abundance	Lysosomal enzymes, α-galactosidase-A (GLA) and lysosomal acid lipase (LIPA)	[89]
MAN1A2	Progressively trim alpha-1,2-linked mannose residues from Man(9)GlcNAc(2) to produce Man(5)GlcNAc(2).
ER mannosidase MAN1B1	Plays an important role in the disposal of misfolded glycoproteins
MAN1C1 and MGAT1 (quadruple and quintuple knockouts based on the triple knockout by Jin and colleagues [89]	Trim alpha-1,2-linked mannose residues from Man(9)GlcNAc(2) to produce first Man(8)GlcNAc(2) then Man(6)GlcNAc and a small amount of Man(5)GlcNAc. Essential for the conversion of high-mannose to hybrid and complex N-glycans	Quadruple and quintuple knockouts	Elimination of all the hybrid-type and complex-type N-glycans with only the high-mannose-type N-glycans present	Lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1)	[86]
Sialyl transferases, ST6GAL1, ST3GAL3 and ST3GAL4 ST6GAL1	Add sialic acid to the terminal portions of the N- or O-linked sugar chains of glycoproteins.	Overexpression Overexpression	Enhanced sialylation Increased α2,6 sialylation	EPO Trastuzumab F243A mutant antibody	[91] [92]
Golgi mannosidases MAN2A1 and MAN2A2	Control conversion of high mannose to complex N-glycans	Double Knockout	Hybrid-type N-glycans only	Lysosomal acid lipase (LIPA) and Fc of immunoglobulin G1 (IgG1)	[88]
Fucosyltransferase 8 (FUT8)	Catalyzes the addition of fucose in alpha 1-6 linkage to the first GlcNAc residue, next to the peptide chains in N-glycans.	Triple knockout with MAN2A1 and MAN2A2	Hybrid-type N-glycans only without core fucosylation	Lysosomal acid lipase (LIPA) and Fc of immunoglobulin G1 (IgG1)	[88]