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21 Introduc�on 1 Glycosyltransferases can be derived from microbial or mammalian sources, each having their own advantages and disadvantages. 113 Expression of mammalian GTs requires expression inmammalianor insect cells, sinceprokaryo�c systemso�en lackglycosyla�on, disulfide bonding and chaperones required for these proteins. Using eukaryo�c cell systems usually limits the scale of mammalian GT expression. 114 Mammalian enzymes are o�en found to be selec�ve to specific acceptor substrates, while bacterial enzymes are usually easier to express in higher yields and have a broader substrate scope. 115 It is hard to find bacterial GTs that synthesize the same glycan linkages as mammalian GTs based on their amino acid sequences. 113 S�ll, bacterial transferases have been iden�fied that form various glycosidic linkages, including those found in globo- and ganglio-series oligosaccharides. The bacterial enzymes LgtC and LgtD were iden�fied to synthesize globo-series Gb3, Gb4 and Gb5 star�ng from lactose. α1,4-Galactosyltransferse LgtC efficiently couples donor substrate UDP-Gal with a lactose acceptor and selec�vely provides Galα1,4Lac (Gb3). 116 This enzyme accepts small subs�tuents at the reducing end of lactose, such as benzyl (Bn). 117 LgtD is a β1,3-N-acetylgalactosaminyltransferase and has been used synthesize Gb4 from Gb3. Furthermore, a bifunc�onal ac�vity was found for LgtD, since it also catalyzes the forma�on of Gb5 from Gb4. 118,109 Another β1,3-galactosyltransferase, CgtB from C. jejuni , has been used to synthesize Gb5 as well, but can cause polygalactosyla�on in case of excess UDP-Gal. 109 Mimics of the ganglio-series have been found in the bacterium C. jejuni and the corresponding GTs have been iden�fied and expressed. 80,109,119,120 Two α2,3- sialyltransferases have been isolated from from C. jejuni and have been used for the synthesis of GM3 (Neu5Acα2,3Lac): Cst-I and Cst-II. Cst-II is a bifunc�onal enzyme since it also has α2,8-sialyltransferase ac�vity. 119 A Pasteurella multocida mutant M144D α2,3-sialyltransferase (PmST1) has frequently been used for α2,3-sialyla�on, including the synthesis of GM3. 109 PmST1 showed poor ac�vity on Lac-Sph, therefore another α2,3-sialyltransferase, PmST3, was used to synthesize GM3-Sph. 121 CgtA is a β1,4-N-acetylgalactosyltransferase from C. jejuni and has been iden�fied and used to synthesize GM2 (GalNAcβ1,4[α2,3Neu5Ac]-Lac) from GM3. 119,120 Aforemen�oned β1,3- galactosyltransferase CgtB was iden�fied and used to catalyze the forma�on of GM1 (Galβ1,3GalNAcβ1,4[α2,3Neu5Ac]Lac) from GM2. 119,120 Besides a suitable sugar nucleo�de donor, glycan acceptor and glycosyltransferase, an op�mal buffer pH and reac�on temperature are required for efficient oligosaccharide synthesis. Furthermore, certain GTs require metal ions (Mg 2+ or Mn 2+ ), which coordinate to the enzyme DXD mo�f and stabilize phosphate groups of the sugar nucleo�de donor. 109,122 Enzyma�cally synthesized oligosaccharides are mainly in their natural (“deprotected”) form and enzyme precipita�on (by ice-cold EtOH), spin filtra�on, Bio- Gel P2 and HILIC are commonly used purifica�on methods. 109 Meanwhile, enzyma�c synthesis of glycolipids requires other purifica�on methods due to the introduc�on of a lipophilic residue. Frequently used methods for glycolipid purifica�ons are C18-based SPE or HPLC. 121