Table 3.

Comparison of glycoengineering methods

	Genetic glycoengineering	Glycosyltransferase glycoengineering	Chemoenzymatic glycoengineering
Strategies	Modify intracellular glycosylation pathways and enzymes via genetic engineering.	Extend monosaccharide residues by glycosyltransferases in vitro.	Modify sugar chains by endoglycosidases and their mutants, together with chemically synthesized active glycan oxazolines.
Methods	Remold sialyltransferases; increase CMP-Neu5Ac-associated enzymes or transporters; inhibit or eliminate sialidases; introduce new N-glycosylation sites.	Construct one-pot system with monosaccharide precursors and glycosyltransferases.	Deglycosylate IgG by an ENGase, prepare oxazoline derivatives as sugars donors via chemical methods, and transglycosylate oxazoline donor to glycoprotein.
Pros	Versatility	Simplicity and relatively purified products.	Simplicity; relatively purified and unlimited products.
Cons	Low efficiency and hybrid glyco-products	Limited glyco-products; difficulty and high cost of active glycan substrates.	Unavoidable hydrolytic activity of ENGase mutant; difficult to achieve oligosaccharide substrates in a large scale.

CMP, cytidine monophosphate; Neu5Ac, N-acetylneuraminic acid; ENGase, endoglycosidase.