Engineering of glycosidases and glycosyltransferases

Engineering of glycosidases and glycosyltransferases Susan M Hancock*, Mark D Vaughan* and Stephen G Withers In recent years, substantial advances have been made in the engineering of glycosidases and glycosyltransferases for the synthesis and degradation of glycan structures. Key developments include improvement of the thermostability of xylanase through comprehensive saturation mutagenesis, creation of the first glycosynthase derived from an inverting glycosidase and the emergence of a new class of modified glycosidases capable of efficiently synthesizing thioglycosidic linkages. Of particular note is the increased use of random mutagenesis and directed evolution tactics for tailoring glycosidase activity. Although the engineering of glycosyltransferases is still in its early stages, recent work on the structure-based alteration of substrate specificity and the manipulation of glycosyltransferase profiles in whole cells to effect complex changes in in vivo glycobiology probably foreshadows a wave of considerable innovation in this area. Addresses Department of Chemistry, University of British Columbia, Rm W300 6174 University Boulevard, Vancouver, British Columbia, V6T 1Z3, Canada * These authors contributed equally to this work. Corresponding author: Withers, Stephen G. ([email protected])

a-D-galactopyranose [UDP-Gal]), to an acceptor. Whereas inverting GTs follow a mechanism analogous to that of inverting glycosidases (Figure 1a), the dearth of evidence for a strict double-displacement mechanism in retaining GTs has prompted the proposal of an SNi-like mechanism [4]. On the basis of sequence similarity, glycosidases and GTs have been classified into approximately 100 and 85 families, respectively. Glycosidases have been found to adopt a plethora of structural folds, whereas, in stark contrast, the majority of GTs have only two structural folds, fold A (GTfA) and B (GTfB). Native glycosidases and GTs have proven extremely useful for in vitro carbohydrate manipulation but, in spite of their huge diversity of sequences, folds and substrate specificities, there are still glycosidic linkages for which a catalyst with the requisite specificity is not known: this is the domain of the protein engineers. This review aims to provide an overview of recent advances in glycosidase and GT engineering. These studies have principally focused on probing mechanism and altering substrate specificities and physical properties of the enzymes, with an emphasis towards the application of engineered catalysts in glycoside synthesis.

Current Opinion in Chemical Biology 2006, 10:509–519 This review comes from a themed issue on Mechanisms Edited by Carol A Fierke and Dan Herschlag Available online 14th August 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.07.015

Introduction In biological systems, glycosyltransferases (GTs) and glycosidases are responsible for the synthesis and catabolism of carbohydrates. Glycosidases hydrolyze glycosidic linkages with net retention or inversion of stereochemistry, using mechanisms that have been extensively studied and reviewed [1–3]. Briefly, inverting glycosidases proceed via a general acid/base-catalyzed direct displacement (Figure 1a), whereas retaining glycosidases use a double-displacement mechanism in which an active site nucleophile attacks the anomeric centre to generate a covalent glycosyl–enzyme intermediate, which is subsequently hydrolyzed in a general acid/base-catalyzed manner (Figure 1b). GTs catalyze the transfer of a monosaccharide from an activated donor, such as a sugar– nucleotide in Leloir GTs (e.g. uridine diphosphate www.sciencedirect.com

Glycosidases The use of enzymes in industrial processes is becoming increasingly widespread because of their catalytic efficiency, their ability to operate under ‘green’ conditions, and their ease of production in large quantities through fermentation procedures [5,6]. The breakdown of glycan structures is of great importance in many processes in the food, pulp and paper, textile, sanitation and agricultural industries. As such, glycosidases have been extensively subjected to protein engineering tactics to improve or alter catalytic activity and substrate specificity, and to modify optimum reaction conditions. A current area of intense interest is the use of glycosidases, primarily cellulases and xylanases, in the conversion of lignocellulosic biomass, which consists of cellulose, hemicellulose (non-cellulose polysaccharides such as glucans, mannans and xylans) and lignin (a complex polymer incorporating numerous phenolic residues), into forms suitable for biofuel production [7,8]. New developments in this area have been explored in recent reviews [9,10]. Random mutagenesis and directed evolution approaches have been used in the generation of altered enzymes for industrial applications [5,6,11,12]. A particularly exhaustive approach to enzyme evolution through random mutagenesis was used to increase the thermostability of a Current Opinion in Chemical Biology 2006, 10:509–519

510 Mechanisms

Figure 1

Catalytic mechanisms for wild-type and engineered glycosidases and GTs.

xylanase [13]. This methodology, dubbed gene site saturation mutagenesis (GSSM), was employed to subject each of the 189 amino acid residues in the protein to saturation mutagenesis to generate a library of modified enzymes, each altered at a single position. This library was then screened for variants with increased thermostability, and nine single amino acid changes were identified that contribute to increased stability. These nine single substitutions were then combinatorially assembled to generate all 512 possible variants. Another round of screening identified eleven enzymes with melting temperatures up to 35 8C higher than that of the wild-type enzyme. Current Opinion in Chemical Biology 2006, 10:509–519

Modified glycosidases for glycoside synthesis Transglycosidases

Glycosidases have emerged as a useful tool in the synthesis of glycosides through transglycosylation reactions, with numerous successful applications in the preparation of diverse glycan structures [14,15]. The utility of glycosidases in synthesis has also benefited from various engineering strategies. A recent report outlines the rational modification of Sulfolobus solfataricus b-glycosidase to accept a wider range of substrates in transglycosylation reactions through the alteration of two key residues involved in substrate recognition, providing access to www.sciencedirect.com

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many different glycoside linkages, including the especially problematic b-mannosyl and b-xylosyl linkages [16]. Directed evolution strategies have been effective in enhancing transglycosidase activity. Through random mutagenesis and in vitro recombination, Dion and coworkers [17] were able to diminish the hydrolytic activity of the Thermus thermophilus b-glycosidase while substantially increasing the transglycosidase activity, therefore enabling the synthesis of b-1,3-linked products in much higher yields than is typically achievable through glycosidase-catalyzed transglycosylation reactions.

activated glycosyl donor to a suitable acceptor through the mechanism shown in Fig. 1c. Since this first development, a number of other glycosynthases have been generated (Table 1) [19–21]. Notable among those recently developed is the glycosynthase derived from the T. thermophilus b-glycosidase, which is capable of synthesizing in high yields the b-1,3 glycosidic linkages found in several antigens and in some plant signalling molecules [22]. Also of interest is the glycosynthase enzyme developed from the Thermotoga maritima b-glucuronidase for the synthesis of b-linked glucuronic and galacturonic acid conjugates [23], which are found in numerous important biological molecules, such as plant and bacterial cell walls and the mammalian glycosaminoglycans, including heparin, heparan sulfate, chondroitin sulfate, and hyaluronan [24]. Additionally, the first glycosynthase derived from a retaining endoxylanase (Cellulomonas fimi endo-b-1,4-xylanase) has been reported, which enables the synthesis of xylo-oligosaccharides consisting of four to twelve residues [25].

Glycosynthases

Although glycosidases have proven to be extremely valuable in carbohydrate syntheses, the use of these enzymes is subject to two key limitations, namely the challenge of driving the reaction in a thermodynamically disfavoured direction and the enzymatic degradation of the reaction product. In the late 1990s, a new class of glycosidase mutants was introduced, which catalyze the synthesis of new glycosidic linkages but do not hydrolyze the newly formed linkages, thereby driving the reaction in the synthetic direction [18]. These mutant glycosidases, termed ‘glycosynthases’, are rendered hydrolytically incompetent through the replacement of the nucleophilic residue with another unable to perform the same function. When supplied with glycosyl fluoride substrates of the opposite anomeric configuration to that of the natural substrate, the enzyme is often able to transfer this

Until very recently, all of these enzymes were derived from b-retaining glycosidases. However, Honda and Kitaoka [26] have reported the first glycosynthase derived from an inverting enzyme, the reducing end xylose-releasing exo-oligoxylanase (Rex) from Bacillus halodurans. Single-site saturation mutagenesis at the aspartate residue that serves as the catalytic base led to

Table 1 Glycosynthases, thioglycoligases and thioglycosynthases to date Parent Glycosidase

Glycoside hydrolase family

Catalytic activity

Altered residue(s)

Linkages synthesized

Endo/exo

Refs

Agrobacterium sp. b-glucosidase (Abg)

GH 1

S. solfataricus b-glucosidase Thermosphaera aggregans b-glycosidase Pyrococcus furiosus b-glucosidase (CelB) T. thermophilus b-glycosidase C. fimi b-mannosidase (Man2A)

GH GH GH GH GH

E. coli b-galactosidase (LacZ) T. maritima b-glucuronidase

GH 2 GH 2

Exo Exo Exo Exo Exo Exo Exo Exo Exo Exo Exo

[18] [33] [36] [74,75] [76] [76] [22] [77] [33] [78] [23]

GH GH GH GH GH GH GH GH GH GH GH

E359 E171 E359/E171 E387 E134 E372 E338 E519 E429 E537 E476 E383 E351 E197 D263 E235 E143 E170 E231 E320 D481 D482 D416

b-1,3/4 b-1,4 b-1,4 b-1,3/4/6 b-1,3/4/6 b-1,3 b-1,3 b-1,3/4 b-1,4 b-1,6 b-1,4

Rhodococcus sp. endoglycoceramidase II H. insolens cellulase B. halodurans reducing-end exo-oligoxylanase C. fimi endo-1,4-b-xylanase Bacillus licheniformis 1,3-1,4-b-glucanase Pyrococcus furiosus laminarinase (LamA) Hordeum vulgare glucan endo-1,3-b-D-glucosidase Cellvibrio japonicus mannanase (Man26A) Schizosaccharomyces pombe a-glucosidase E. coli a-xylosidase (YicI) S. solfataricus a-glucosidase

Glycosynthase Thioglycoligase Thioglycosynthase Glycosynthase Glycosynthase Glycosynthase Glycosynthase Glycosynthase Thioglycoligase Glycosynthase Glycosynthase Thioglycoligase Glycosynthase Glycosynthase Glycosynthase a Glycosynthase Glycosynthase Glycosynthase Glycosynthase Glycosynthase Glycosynthase Thioglycoligase Thioglycoligase

b-1,1 b-1,4 b-1,4 b 1,4 b-1,3/4 b-1,3/4 b-1,3 b-1,4 a-1,4 a-1,4/6 a-1,4

Endo Endo Endo Endo Endo Endo Endo Endo Exo Exo Exo

[28] [79] [26] [25] [80,81] [82] [83,84] [85] [86] [35] [35]

a

1 1 1 1 2

5 7 8 10 16 16 17 26 31 31 31

Derived from an inverting glycosidase. All other enzymes in this table are generated from retaining glycosidases.

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the discovery of nine amino acid substitutions, which confer glycosynthase activity to this enzyme. Among the altered enzymes able to catalyze the glycosynthase reaction, the cysteine-substituted species displays the highest yield of transglycosylation product, an unusual observation given that this substitution has never proven to be the most effective replacement in a glycosynthase derived from a retaining enzyme. Recent work has also expanded the repertoire of glycosides accessible through the glycosynthase chemistry to include glycosphingolipids, a class of therapeutically valuable compounds that have been extremely difficult to access on a larger scale through chemical and chemoenzymatic methods. The need for methods to efficiently access these compounds prompted the design of a glycosynthase derived from Rhodococcus sp. endoglycoceramidase II, a b-endoglycosidase that cleaves the glycosidic linkage between the glycan and lipid moieties of glycosphingolipids [27]. Substitution of the nucleophilic residue in this enzyme with catalytically incompetent replacements generates an enzyme capable of condensing sphingolipids with a series of oligosaccharyl fluorides to generate several lyso-glycosphingolipids, including the GM1 and GM3 gangliosides, in yields up to 95% [28]. Directed evolution tactics are gaining prominence in the improvement and alteration of the catalytic activities of glycosynthases. Random mutagenesis in conjunction with on-plate in vivo screening has served to improve the catalytic activity and expand the substrate promiscuity of the Abg glycosynthase [29]. An initial 50-fold increase in activity achieved through saturation mutagenesis of the nucleophile was followed by two rounds of random mutagenesis and screening to give a further 27fold increase in catalytic efficiency, such that the kinetic parameters for glycoside synthesis by glycosynthase are remarkably similar to those for glycoside hydrolysis by the wild-type enzyme. This improved glycosynthase is also able to efficiently use a-xylosyl fluoride as a glycosyl donor, an interesting observation given that the wild-type enzyme and the original single-mutant Abg glycosynthase catalyze the transfer of xylosyl moieties only very slowly [30]. Further exploration of this ability to transfer the xylosyl moiety revealed that when aryl xylosides are used as acceptors with xylosyl fluoride donors, the transfer occurs at the 3-position, rather than the 4-position as is the case for transfer to aryl glucosides [31]. With an aryl xylobioside as the acceptor, however, transfer occurs at the 4-hydroxyl group, suggesting that monosaccharide and disaccharide bind to the active site of this glycosynthase enzyme in different orientations. An alternative approach for the directed evolution of glycosynthase activity has been reported by Cornish and co-workers [32], who used ‘chemical complementation’ to select for improved activity of the Humicola Current Opinion in Chemical Biology 2006, 10:509–519

insolens Cel7B glycosynthase in vivo through a yeast three-hybrid assay. Briefly, this methodology involves the chemical conjugation of the glycosyl fluoride donor and glycosyl acceptor with methotrexate and dexamethasone, respectively. A reporter gene (in this case LEU2, required for the production of leucine) is placed under the control of a promoter, requiring a transcription activator (consisting of an activation domain and a DNA-binding domain) in a leucine auxotrophic host. The activation and DNA-binding domains of the transcription activator are separately fused to a glucocorticoid receptor and dihydrofolate reductase, respectively. In the presence of active glycosynthase, the glycosyl donor and acceptor are joined, thereby chemically connecting the methotrexate and dexamethasone through a glycoside linker. Interaction of the glucocorticoid receptor and dihydrofolate reductase with these ligands effects the reconstitution of the activator, enabling the expression of the LEU2 gene and allowing growth in the absence of leucine. Using this approach, a fivefold increase in activity was achieved for the Cel7B glycosynthase.

Thioglycoligases and thioglycosynthases A new class of glycosidase mutants capable of catalyzing the efficient synthesis of thioglycosidic linkages was recently reported [33]. These thioglycoligases are generated through substitution of the catalytic acid/base residue in retaining glycosidases with a residue unable to serve in this capacity. When an activated aryl glycoside is used as a glycosyl donor, the glycosyl–enzyme intermediate is still formed and a thiol acceptor, which does not require base catalysis, reacts efficiently with this intermediate to generate the thioglycoside (Figure 1d). Furthermore, the resistance of thioglycosidic linkages to cleavage by glycosidases negates the loss of product through the residual hydrolytic activity of the modified glycosidase. Site-saturation mutagenesis at the acid/base position of Abg revealed that thioglycoligase activity is enhanced when glutamine serves as the replacement for the native glutamate residue, increasing the overall catalytic efficiency by fivefold relative to the initial alanine mutant [34]. Moreover, this improved thioglycoligase is able to use donor sugars with relatively poor leaving groups, such as azide or p-nitrophenol, at efficiencies approximately 100-fold greater than those observed for the alanine-substituted enzyme. Several other thioglycoligases have been generated from diverse enzymes such as the T. maritima b-glucuronidase [23] discussed above and the C. fimi b-mannosidase [33] (Table 1). Additionally, the usefulness of the thioglycoligases was recently enhanced by the generation of enzymes capable of synthesizing a-thioglycosidic linkages, derived from the Family 31 S. solfataricus a-glucosidase and the Escherichia coli a-xylosidase [35]. This technology was further extended with the advent of thioglycosynthases, double-mutant enzymes in which www.sciencedirect.com

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both the catalytic nucleophile and acid/base residues are replaced by residues unable to perform these functions [36]. By using activated glycosyl fluoride donor sugars along with nucleophilic thiosugars, these modified enzymes are able to catalyze the condensation of these two activated species, essentially by serving as a scaffold that orients the reacting functional groups in a productive attack complex (Figure 1e). Remarkably, the Abg thioglycosynthase catalyzes the formation of the thioglycosidic linkage with a kcat value of 9 min 1 (compared with values of 200 min 1 and 50 min 1 for the original single-mutant glycosynthase and thioglycoligase, respectively), despite the absence of the key catalytic residues.

Glycosyltransferases As Nature’s solution to the assembly of glycosidic bonds, GTs, and thus engineered GTs with broadened or requisite substrate specificities, have enormous potential for the synthesis of novel, non-natural and biologically relevant carbohydrate structures, either by the synthesis of non-natural linkages or by the incorporation of non-natural monosaccharides. The difficulty of synthesizing the necessary non-natural nucleotide sugars is being overcome by anomeric kinase and nucleotidyl transferase rational design and directed evolution, which have produced enzymes that process variously configured D- or L-hexose sugars and tolerate diverse sugar modifications [37–39]. At this point, two possible strategies exist for the use of GTs in the synthesis of glycosides containing nonnatural sugars. First, promiscuous GTs might be available that catalyze the requisite non-natural sugar transfer; second, a pre-existing GT substrate specificity might be modified to accept the non-natural substrate using protein engineering approaches. To date, engineering in GTfA has focused almost entirely on donor specificity, a probable consequence of the acceptor binding site only being formed upon donor

binding, complicating crystallographic analysis of putative mutation sites [40]. With over 100 X-ray crystal structures of GTfAs in the protein database, structurebased rational redesign is feasible, as illustrated with the following examples. Crystallographic analysis of the bovine b-1,4-galactosyltransferase suggested that steric clashes between residue E317 and the equatorial C-4 hydroxyl of glucose (Glc) were responsible for the differentiation between UDPGal and UDP-Glc substrates. Upon mutation of a neighbouring residue (R228K), the E317 residue was relocated, creating adequate space for the equatorial 4-hydroxyl of UDP-Glc (Figure 2a). Accordingly, the R228K mutation improved glucosyltransferase (GlcT) activity by 15-fold at the expense of galactosyltransferase (GalT) activity, which was reduced by sixfold [41]. In another study, the Y289L mutation of GalT rendered the enzyme capable of transferring both N-acetyl-2-amino-2-deoxy-Dgalactopyranose (GalNAc) and Gal at comparable rates [42]. This was in concordance with the X-ray crystal structure, which predicted that such a mutation should provide space for the more sterically demanding C-2 Nacetyl group (Figure 2b). The enhanced tolerance of the GalT Y289L mutant to C-2 donor sugar substituents was subsequently exploited to transfer non-natural 2-keto sugars to O-N-acetylglucosamine (O-GlcNAc)-modified proteins [43]. This introduced ketone was then used as a functional handle for labelling, and provided a novel, efficient method for detecting O-GlcNAc post-translational modifications. Computational modelling of UDP-Glc into the crystal structure of human b-1,3-glucuronosyltransferase I suggested that a single residue was crucial for recognition of glucuronic acid (GlcA). By mutating this residue (H308R), the authors created a broad specificity enzyme that could use UDP-Glc, UDP-mannose and

Figure 2

Crystallographic analysis of bovine b-1,4-galactosyltransferase (GalT) [41,42]. (a) Overlap of wild-type (PDB: 1O0R, gold) and R228K GalT (PDB: 1YRO, grey) with UDP-Gal substrate (yellow) and Mn2+ (cyan) highlighting the relocation of D318 upon R228K mutation, and the interactions between Oe1 of E317 wild-type GalT and R228 NH1 (green dashes, 3.1 A˚), and R228K NZ and D318 Od1/2 of the R228K GalT mutant (red dashes, 2.9 and 3.0 A˚). (b) Wild-type GalT (PDB 1OQM, grey) showing the close interaction between Y289 and the 2-N-acetyl of UDP-GalNAc (yellow). www.sciencedirect.com

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UDP-GlcNAc donor sugars with 13–42% efficiency relative to wild-type UDP-GlcA activity [44].

at this position along with the tyrosine hydroxyl group for optimal activity [53].

Substrate specificity engineering has also been conducted on the cytotoxins of Clostridia species, which are GTs that glycosylate and thereby inactivate Rho family GTPases. The crystal structure of the Clostridium difficile toxin B, a GlcT, together with sequence alignments with Clostridium novyi a-toxin, a N-acetylglucosaminyltransferase (GlcNAcT), identified that residues I/S383 and Q/A385 were responsible for the observed differences in donor specificities. By exchanging these residues, it was possible to improve the kcat/KM of toxin B towards GlcNAc donor by 8000-fold as a result of improved UDP-GlcNAc binding, while reducing GlcT activity by 75%. Correspondingly, the opposite mutations in a-toxin enhanced GlcT activity 360-fold and simultaneously eliminated GlcNAcT activity [45].

Activity knock-out is another engineering approach which has been used on a hyaluronan synthase from Pasteurella multocida to synthesize glycosaminoglycans on the solid phase [54,55]. Hyaluronan synthase is naturally a dual-action enzyme that possesses two catalytic domains with b-1,3-N-acetylglucosaminyltransferase and b-1,4-glucuronosyltransferase activities, respectively. Mutation of the nucleotide binding (DxD) motif of one domain selectively eliminated activity and generated monofunctional GTs that could be immobilized and used in alternation for the production of monodisperse, structurally defined glycosaminoglycans [56,57].

The GTs responsible for the synthesis of human blood group antigens A and B (a-1,3-N-acetylgalactosaminyltransferase and a-1,3-galactosyltransferase, respectively) were long ago shown to differ by only four amino acids (R/ G176, G/S235, L/M266 and G/A268), thus substitution of these four residues in both these transferases resulted in switching of their GT activities [46]. Subsequently, the contribution of each of these residues to specificity was elaborated in an elegant combination of mutagenesis, kinetic and crystallographic analysis [47–50]. More recently, an a-1,3-galactosyltransferase mutant (P234S) was identified from a rare cis-AB allele that produces both A and B blood group antigens. The recombinant P234S mutant was shown to have a completely reversed donor specificity from UDP-Gal to UDP-GalNAc (in spite of being bifunctional in vivo), even though the four ‘crucial’ amino acids associated with B-antigen specificity were still intact [51]. X-ray crystallographic analysis revealed that an alternative conformation of M266 was induced by the P324S mutation, resulting in more space for the GalNAc 2-N-acetyl group. These examples of engineering in blood group antigen-synthesizing GTs imply that similar enzyme specificities can be attained through many differing mutagenic routes, a fact that makes sense from an evolutionary perspective and is extremely encouraging for the protein engineer. The engineering of GTs has not been limited to the introduction of point mutations. Domain-swapping between different Helicobacter pylori strains has been employed in an attempt to better understand the regioselectivity of a-1,3/4-fucosyltransferases. Twelve fucosyltransferase chimeras were generated, from which residues 347–353 were identified as the key region that abolished or conferred a-1,4 activity [52]. Alanine scanning mutagenesis of these seven amino acids revealed that Y350 was solely responsible for a-1,4 activity, and further mutagenesis confirmed the absolute need for an aromatic residue Current Opinion in Chemical Biology 2006, 10:509–519

An engineering approach was used with the a-galactosyltransferase from Neisseria meningitidis in an attempt to trap a reaction intermediate and thereby delineate the normal mechanism of a retaining transferase [58]. Mutation of Gln189, a residue that was shown by crystallography to be tantalizingly poised for nucleophilic attack on the substrate anomeric centre, to a more nucleophilic glutamate residue did indeed result in trapping of a covalent glycosyl enzyme species. This covalent species was turned over with a similar rate constant to the kcat for this mutant. Unfortunately, peptide mapping revealed that the labelled residue was in fact Asp190, a residue located approximately 9 A˚ away from the active site, according to a crystal structure of the Q189E mutant with bound UDP2-deoxy-2-fluoro-Gal. Although these results did little to further our understanding of retaining GT mechanism, they highlighted the importance of using biochemical studies in conjunction with crystallography to determine the catalytic relevance of trapped species. In comparison with GTfA, examples of engineering in GTfB are rare because of fewer crystallographic analyses (19 as of July 2006), and as a result rational engineering attempts are largely based on sequence homology. Nevertheless, GTfBs are promising prototypes for GT engineering because of their natural propensity to glycosylate chemically diverse non-sugar acceptors. In a classic example of GTfB engineering, GTs involved in the biosynthesis of urdamycin were mutated at the substrate specificity region to produce enzymes that created novel b-1,4 linkages between 12b-derhodinosyl-urdamycin G and the C4-hydroxy group of D-olivose [59,60]. More recently, a single residue (corresponding to Q382 in GlcT, and H374 in GalT) that conferred donor specificity differences between plant GlcTs and GalTs from GTfB was identified by sequence homology. The H374Q mutation in UDP-galactose:anthocyanin 3-O-galactosyltransferase from Aralia cordata resulted in a 40-fold increase in UDP-Glc binding with little change in Vmax, while also preserving its native GalT activity; however, the Q382H mutation in UDP-Glc:flavonoid 7-O-glucosyltransferase www.sciencedirect.com

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from Scutellaria baicalensis did not introduce GalT activity and crippled GlcT activity [61]. The single amino acid mutations of GTs discussed above have been effectively used to alter donor substrate specificity. Interestingly, the outcomes of each experiment are distinct: mutations of the human blood group antigensynthesizing GTs created novel and specific activities [46,51], whereas in human b-1,3-glucuronosyltransferase a promiscuous enzyme was produced [44]. Even if, to date, the rational enzyme engineer does not possess the necessary knowledge to premeditate these effects, selection against (or for) enzyme promiscuity could be carried out by directed evolution experiments, using methods analogous to those preventing promiscuity in directed evolution of aminotransferase (JF Kirsch and S Sivaraman, personal communication). Because of the conservation of GT structure, it has been suggested that manipulation of GTs might be simpler than previously envisioned [62], although at present GT engineering is still in its infancy in comparison with glycosidase engineering examples. As yet, there are no examples of GT directed evolution, but this is likely to be addressed

shortly (see Update), particularly because directed evolution does not require knowledge of the enzyme structure. Indeed, the recent creation of a novel high-throughput screening system for GT libraries [63] is an important step in the right direction, even though its utility is yet to be demonstrated. Although this review largely reports progress in engineering of glycosidases and GTs by sequence changes, some interesting examples exist whereby whole cells are engineered by introducing genes for carbohydrate-processing enzymes, examples of which also show the valuable biosynthetic capabilities of these enzymes in the largescale preparation of synthetically challenging oligosaccharide structures. These glycosylation reactions are performed by whole cells overexpressing the genes encoding the appropriate glycosidases, GTs and sugar–nucleotide biosynthetic machinery. Metabolically engineered E. coli have been used to synthesize the oligosaccharide moieties of gangliosides GM3, GM2 [64], GD3 [65] and GM1 [66] (Figure 3), the Lewis x epitope [67] and the blood group H-antigen [68]. This work was further developed in attempts to humanize the glycosylation pathway of

Figure 3

Metabolically engineered pathway towards the oligosaccharide moieties of gangliosides GM3, GM2 and GM1 in E. coli K12. Lactose Gal(b1,4)Glc and N-acetylneuraminic acid (Neu5Ac) are internalized by specific permeases LacY and NanT, and cannot be degraded because of b-galactosidase (LacZ) and aldolase (NanA) gene deletions. A series of plasmid-encoded enzyme reactions then follow. The nucleotide-activated form of Neu5Ac (CMP-Neu5Ac) is created in situ by CMP-Neu5Ac synthase, and is subsequently transferred to lactose to form sialyllactose (GM3 oligosaccharide) by a-2,3-sialyltransferase (Lst). UDP-GalNAc, formed from UDP-Glc by UDP-GlcNAc C4 epimerase (WbpP) allows the b-1,4-GalNAc transferase (CgtA) to catalyze the formation of the GM2 oligosaccharide, which then acts as an acceptor for b-1,4-galactosyltransferase (CgtB) to give the GM1 oligosaccharide. Enzymes are in bold; UDP, uridine diphosphate; CTP, cytidine triphosphate; PPi, inorganic pyrophosphate. www.sciencedirect.com

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Pichia pastoris [69,70,71]. To secrete glycoproteins with homogenous complex N-glycosylation, chimeric GTs made from the fusion of various GT catalytic domains and leader sequences were screened for activity and correct localization in yeast cells, in which the endogenous yeast high mannose type N-glycan glycosylation pathway had been knocked out. Further extension of this approach through the engineering of mammalian cells represented a massive step towards the clinical reality of xenotransplantation. A point mutation of a1,3-galactosyltransferase produced pigs that were deficient in the synthesis of the Gala-1,3-Gal epitope, widely believed to be the cause of hyperacute rejection in pig-toprimate xenografts [72]. In a similar manner, reducing this Gala-1,3-Gal epitope on mouse-derived retroviral vectors prevented recognition by the human immune system, thus producing serum-stabilized retroviral vectors for gene therapy. In this case, however, instead of knocking out a-galactosyltransferase activities, the catalytic domain of human a-2,3-sialyltransferase was fused with Golgi signal sequences for compartment-specific localization, creating chimeric GTs which competed with the murine a-1,3-galactosyltransferase [73].

Conclusions The need for efficient synthetic routes to glycoconjugates has stimulated significant advances in the engineering of glycosidases and GTs. Glycosidases have been engineered to modify their substrate specificities and physical properties, and to produce enzymes with novel mechanisms, either by using the wealth of available structural information for rational design, or implementing directed evolution strategies with powerful new screening approaches, such as the discussed yeast three-hybrid system. Although rational GT engineering attempts remain fairly simple, reprogramming mammalian, eukaryotic and prokaryotic cells by introducing GT and glycosidase genes has been extremely successful, producing efficient ‘glycosylation factories’. The large research investment for a better understanding of the fundamental mechanisms of glycosidases has paid dividends in the ability to engineer these enzymes, a lesson which will hopefully be duplicated for GTs.

Update A novel, truly high-throughput assay for screening GT mutants utilizing fluorescence-activated cell sorting (FACS) was very recently described. Using this approach, a library of 106–107 sialyltransferase mutants was rapidly sorted and a single point mutation was identified that increased transfer activity up to 400-fold [87].

Acknowledgements The authors would like to thank the Natural Sciences and Engineering Research Council of Canada, the Protein Engineering Network of Centres of Excellence of Canada, the Canadian Institutes of Health Research, Neose Technologies Inc. (Horsham, PA, USA) and the Royal Society (UK) for financial support. Current Opinion in Chemical Biology 2006, 10:509–519

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20. Perugino G, Trincone A, Rossi M, Moracci M: Oligosaccharide synthesis by glycosynthases. Trends Biotechnol 2004, 22:31-37. 21. Perugino G, Cobucci-Ponzano B, Rossi M, Moracci M: Recent advances in the oligosaccharide synthesis promoted by catalytically engineered glycosidases. Adv Synth Catal 2005, 347:941-950. 22. Drone J, Feng HY, Tellier C, Hoffmann L, Tran V, Rabiller C, Dion M: Thermus thermophilus glycosynthases for the efficient synthesis of galactosyl and glucosyl b-1,3-glycosides. Eur J Org Chem 2005:1977-1983. 23. Mullegger J, Chen H, Chan WY, Reid SP, Jahn M, Warren RAJ, Salleh HM, Withers SG: Thermostable glycosynthases and thioglycoligases derived from Thermotoga maritima b-glucuronidase. ChemBioChem 2006, 7:1028-1030. 24. Whitelock JM, Iozzo RV: Heparan sulfate: a complex polymer charged with biological activity. Chem Rev 2005, 105:2745-2764. 25. Kim Y-W, Fox DT, Hekmat O, Kantner T, McIntosh LP, Withers SG: Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi. Org Biomol Chem 2006, 4:2025-2032. 26. Honda Y, Kitaoka M: The first glycosynthase derived from  an inverting glycoside hydrolase. J Biol Chem 2006, 281:1426-1431. Before this publication, all glycosynthases had been derived from retaining glycosidases. These authors postulated that glycosynthesis could be achieved through a reaction pathway similar to the Hehre resynthesis mechanism, in which glycosyl fluorides of opposite anomeric configuration relative to the cleaved linkage could serve as donors in transglycosylation reactions. It was previously known that removal of the catalytic base would shut down the hydrolysis of the transglycosylation product. Interestingly, they found that cysteine served as the most effective replacement for the catalytic base in the glycosynthase. 27. Ito M, Yamagata T: A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide in neutral and acidic glycosphingolipids. J Biol Chem 1986, 261:14278-14282. 28. Vaughan MD, Johnson K, DeFrees S, Tang X, Warren RAJ, Withers SG: Glycosynthase-mediated synthesis of glycosphingolipids. J Am Chem Soc 2006, 128:6300-6301. 29. Kim YW, Lee SS, Warren RA, Withers SG: Directed evolution of a  glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J Biol Chem 2004, 279:42787-42793. A very simple on-plate screening approach was used in combination with random mutagenesis approaches to improve the catalytic parameters of the Abg glycosynthase to levels approaching those of the wild-type enzyme. In addition to the catalytic nucleophile, three other mutations were identified in the optimized enzyme. The altered residues do not directly interact with the substrates, but appear to exert their effects through longer range structural effects that perturb the structure of the active site. 30. Kempton JB, Withers SG: Mechanism of Agrobacterium b-glucosidase: kinetic studies. Biochemistry 1992, 31:9961-9969. 31. Kim YW, Chen H, Withers SG: Enzymatic transglycosylation of xylose using a glycosynthase. Carbohydr Res 2005, 340:2735-2741. 32. Lin H, Tao H, Cornish VW: Directed evolution of a glycosynthase  via chemical complementation. J Am Chem Soc 2004, 126:15051-15059. The chemical complementation methodology is well-suited to the evolution of enzymes involved in condensation reactions. By coupling the donor and acceptor to small-molecule ligands of dihydrofolate reductase and the glucocorticoid receptor (which are, in turn, fused to the DNAbinding and activation domains of a transcription activator), the authors were able to correlate enzyme efficiency with the rescue of a lethal mutation, achieving a fivefold improvement in Cel7B glycosynthase activity. 33. Jahn M, Marles J, Warren RA, Withers SG: Thioglycoligases: mutant glycosidases for thioglycoside synthesis. Angew Chem Int Ed Engl 2003, 42:352-354. www.sciencedirect.com

34. Mullegger J, Jahn M, Chen HM, Warren RA, Withers SG: Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts. Protein Eng Des Sel 2005, 18:33-40. 35. Kim YW, Lovering A, Chen H, Kantner T, McIntosh LP, Strynadka NCJ, Withers SG: Expanding the thioglycoligase strategy to the synthesis of a-linked thioglycosides allows structural investigation of the parent enzyme/substrate complex. J Am Chem Soc 2006, 128:2202-2203. 36. Jahn M, Withers SG: Thioglycosynthases: double mutant  glycosidases that serve as scaffolds for thioglycoside synthesis. Chem Commun (Camb) 2004:274-275. This work is a combination of the thioglycoligase and glycosynthase methodologies, enabling the utilization of glycosyl fluoride donors (the same substrates as employed in the glycosynthase reactions, rather than the nitrophenyl glycosides normally used in thioglycoligase reactions) for the enzyme-catalyzed synthesis of thioglycosides. 37. Yang J, Liu L, Thorson JS: Structure-based enhancement of the first anomeric glucokinase. ChemBioChem 2004, 5:992-996. 38. Hoffmeister D, Yang J, Liu L, Thorson JS: Creation of the first D/L-sugar kinases by means of directed evolution. Proc Natl Acad Sci USA 2003, 100:13184-13189. 39. Barton WA, Biggins JB, Jiang J, Thorson JS, Nikolov DB: Expanding pyrimidine diphosphosugar libraries via structurebased nucleotidyltransferase engineering. Proc Natl Acad Sci USA 2002, 99:13397-13402. 40. Qasba PK, Ramakrishnan B, Boeggeman E: Substrate-induced conformational changes in glycosyltransferases. Trends Biochem Sci 2005, 30:53-60. 41. Ramakrishnan B, Boeggeman E, Qasba PK: Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine b-1,4-galactosyltransferase I. Biochemistry 2005, 44:3202-3210. 42. Ramakrishnan B, Qasba PK: Structure-based design of b-1,4-galactosyltransferase I (b4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity. J Biol Chem 2002, 277:20833-20839. 43. Khidekel N, Arndt S, Lamarre-Vincent N, Lippert A,  Poulin-Kerstien KG, Ramakrishnan B, Qasba PK, Hsieh-Wilson LC: A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J Am Chem Soc 2003, 125:16162-16163. A novel detection method for O-GlcNAc modified proteins is reported which uses a mutant of bovine b-1,4-galactosyltransferase. The increased C-2 substituent tolerance of the Y289L mutant enables the transfer of a non-natural 2-keto sugar, which is used as a chemical handle for labelling and detection. 44. Ouzzine M, Gulberti S, Levoin N, Netter P, Magdalou J, Fournel-Gigleux S: The donor substrate specificity of the human b-1,3-glucuronosyltransferase I toward UDPglucuronic acid is determined by two crucial histidine and arginine residues. J Biol Chem 2002, 277:25439-25445. 45. Jank T, Reinert DJ, Giesemann T, Schulz GE, Aktories K: Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis. J Biol Chem 2005, 280:37833-37838. 46. Yamamoto F, Clausen H, White T, Marken J, Hakomori S: Molecular genetic-basis of the histo-blood group ABO system. Nature 1990, 345:229-233. 47. Lee HJ, Barry CH, Borisova SN, Seto NOL, Zheng RB, Blancher A, Evans SV, Palcic MM: Structural basis for the inactivity of human blood group O2 glycosyltransferase. J Biol Chem 2005, 280:525-529. 48. Palcic MM, Seto NOL, Hindsgaul O: Natural and recombinant A and B gene encoded glycosyltransferases. Transfus Med 2001, 11:315-323. 49. Patenaude SI, Seto NOL, Borisova SN, Szpacenko A, Marcus SL, Palcic MM, Evans SV: The structural basis for specificity in human ABO(H) blood group biosynthesis. Nat Struct Biol 2002, 9:685-690. Current Opinion in Chemical Biology 2006, 10:509–519

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50. Seto NOL, Palcic MM, Compston CA, Li H, Bundle DR, Narang SA: Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes. J Biol Chem 1997, 272:14133-14138. 51. Marcus SL, Polakowski R, Seto NOL, Leinala E, Borisova S, Blancher A, Roubinet F, Evans SV, Palcic MM: A single point mutation reverses the donor specificity of human blood group B-synthesizing galactosyltransferase. J Biol Chem 2003, 278:12403-12405. 52. Ma B, Wang G, Palcic MM, Hazes B, Taylor DE: C-terminal amino acids of Helicobacter pylori a-1,3/4 fucosyltransferases determine type I and type II transfer. J Biol Chem 2003, 278:21893-21900. 53. Ma B, Lau LH, Palcic MM, Hazes B, Taylor DE: A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori a1,3/4 fucosyltransferase determines substrate specificity. J Biol Chem 2005, 280:36848-36856. 54. Jing W, DeAngelis PL: Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem 2004, 279:42345-42349. 55. DeAngelis PL, Oatman LC, Gay DF: Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors. J Biol Chem 2003, 278:35199-35203. 56. Jing W, DeAngelis PL: Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida. Glycobiology 2003, 13:661-671. 57. Jing W, DeAngelis PL: Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide. Glycobiology 2000, 10:883-889. 58. Lairson LL, Chiu CPC, Ly HD, He S, Wakarchuk WW, Strynadka NCJ, Withers SG: Intermediate trapping on a mutant retaining a-galactosyltransferase identifies an unexpected aspartate residue. J Biol Chem 2004, 279:28339-28344. 59. Hoffmeister D, Wilkinson B, Foster G, Sidebottom PJ, Ichinose K, Bechtold A: Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity. Chem Biol 2002, 9:287-295. 60. Hoffmeister D, Ichinose K, Bechtold A: Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity. Chem Biol 2001, 8:557-567. 61. Kubo A, Arai Y, Nagashima S, Yoshikawa T: Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation. Arch Biochem Biophys 2004, 429:198-203. 62. Hu Y, Walker S: Remarkable structural similarities between diverse glycosyltransferases. Chem Biol 2002, 9:1287-1296. 63. Love KR, Swoboda JG, Noren CJ, Walker S: Enabling glycosyltransferase evolution: a facile substrate-attachment strategy for phage-display enzyme evolution. ChemBioChem 2006, 7:753-756. 64. Fort S, Birikaki L, Dubois M-P, Antoine T, Samain E, Driguez H: Biosynthesis of conjugatable saccharidic moieties of GM2 and GM3 gangliosides by engineered E. coli. Chem Commun (Camb) 2005:2558-2560. 65. Antoine T, Heyraud A, Bosso C, Samain E: Highly efficient biosynthesis of the oligosaccharide moiety of the GD3 ganglioside by using metabolically engineered Escherichia coli. Angew Chem Int Ed Engl 2005, 44:1350-1352. 66. Antoine T, Priem B, Heyraud A, Greffe L, Gilbert M,  Wakarchuk WW, Lam JS, Samain E: Large-scale in vivo synthesis of the carbohydrate moieties of gangliosides GM1 and GM2 by metabolically engineered Escherichia coli. ChemBioChem 2003, 4:406-412. Metabolic engineering of E. coli is used to synthesize the oligosaccharidic moieties of gangliosides GM3, GM2 and GM1 in 1 gL 1 yields. Genes for CMP-sialic acid synthase and a-2,3-sialyltransferase from N. meningitiCurrent Opinion in Chemical Biology 2006, 10:509–519

dis, UDP-N-acetylglucosaminyl epimerase from P. aeruginosa and b-1,3galactosyltransferase and b-1,4-N-acetylgalactosaminyltransferase from C. jejuni were introduced, while the galactosidase and aldolase activities were knocked out to prevent substrate degradation. 67. Dumon C, Bosso C, Utille JP, Heyraud A, Samain E: Production of  Lewis x tetrasaccharides by metabolically engineered Escherichia coli. ChemBioChem 2006, 7:359-365. By introducing the genes for rhizobial chitin-synthase and Bacillus circulans chitinase A1, E. coli cells were produced that synthesized chitobiose. The further incorporation of the genes for H. pylori a-1,3fucosyltransferase, and b-1,4-galactosyltransferase and b-1,3-N-acetylglucosaminyltransferase from N. meningitidis enabled the bacteria to generate the synthetically challenging trisaccharidic Lewis x motif (Galb1,4(Fuca1,3)GlcNAc) in yields of 0.6–1.8 gL 1 of culture. 68. Drouillard S, Driguez H, Samain E: Large-scale synthesis of  H-antigen oligosaccharides by expressing Helicobacter pylori a1,2-fucosyltransferase in metabolically engineered Escherichia coli cells. Angew Chem Int Ed Engl 2006, 45:1778-1780. This paper reports the synthesis of the H-antigen via the glycosylation of lactose by b-1,4-galactosyltransferase and b-1,3-N-acetylglucosaminyltransferase from N. meningitidis and H. pylori a-1,2-fucosyltransferase in engineered E. coli cells that also overproduced GDP-fucose. The Hantigen has anti-infective properties, but pharmaceutical application has been limited by the lack of reliable methods for large-scale synthesis, a problem which the authors believe they have now addressed. 69. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H,  Mitchell T, Nett JH, Rausch S, Stadheim TA, Wischnewski H et al.: Production of complex human glycoproteins in yeast. Science 2003, 301:1244-1246. Metabolic engineering of P. pastoris to produce the human N-glycan, GlcNAc2Man3GlcNAc2, is reported. This involved eliminating the endogenous yeast glycosylation pathways and properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyltransferases I and II, and the UDP-GlcNAc transporter. 70. Choi B-K, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU: Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 2003, 100:5022-5027. 71. Bobrowicz P, Davidson RC, Li H, Potgeiter TI, Nett JH, Hamilton SR, Stadheim TA, Miele RG, Bobrowicz B, Mitchell T et al.: Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 2004, 14:757-766. 72. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen S-H, Ball S, Specht SM, Polejaeva IA, Monahan JA et al.: Production of a1,3-galactosyltransferase-deficient pigs. Science 2003, 299:411-414. 73. Hansen W, Grabenhorst E, Nimtz M, Muller K, Conradt HS,  Wirth M: Generation of serum-stabilised retroviruses: reduction of a1,3gal-epitope synthesis in a murine NIH3T3derived packaging cell line by expression of chimeric glycosyltransferases. Metab Eng 2005, 7:221-228. Competition between the native a-1,3-galactosyltransferase and an introduced human sialyltransferase is used to reduce the Gala1,3Gal epitope formation in a mouse-derived packaging cell line. Retroviral vectors produced by these cells showed a 3.5-fold increase in human serum stability. 74. Moracci M, Trincone A, Perugino G, Ciaramella M, Rossi M: Restoration of the activity of active-site mutants of the hyperthermophilic b-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles. Biochemistry 1998, 37:17262-17270. 75. Trincone A, Perugino G, Rossi M, Moracci M: A novel thermophilic glycosynthase that effects branching glycosylation. Bioorg Med Chem Lett 2000, 10:365-368. 76. Perugino G, Trincone A, Giordano A, van der Oost J, Kaper T, Rossi M, Moracci M: Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH. Biochemistry 2003, 42:8484-8493. 77. Nashiru O, Zechel DL, Stoll D, Mohammadzadeh T, Warren RA, Withers SG: b-Mannosynthase: synthesis of b-mannosides www.sciencedirect.com

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with a mutant b-mannosidase. Angew Chem Int Ed Engl 2001, 40:417-420. 78. Jakeman DL, Withers SG: On expanding the repertoire of glycosynthases: mutant b-galactosidases forming b-(1,6)linkages. Can J Chem 2002, 80:866-870. 79. Fort S, Boyer V, Greffe L, Davies GJ, Moroz O, Christianses L, Schulien M, Cottaz S, Driguez H: Highly efficient synthesis of (1 ! 4)-oligo- and poly-saccharides using a mutant cellulase. J Am Chem Soc 2000, 122:5429-5437. 80. Malet C, Planas A: From b-glucanase to b-glucansynthase: glycosyl transfer to a-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile. FEBS Lett 1998, 440:208-212. 81. Fairweather JK, Faijes M, Driguez H, Planas A: Specificity studies of Bacillus 1,3-1,4-b-glucanases and application to glycosynthase-catalyzed transglycosylation. Chem Bio Chem 2002, 3:866-873. 82. van Lieshout J, Faijes M, Nieto J, van der Oost J, Planas A: Hydrolase and glycosynthase activity of endo-1,3-bglucanase from the thermophile Pyrococcus furiosus. Archaea 2004, 1:285-292. 83. Hrmova M, Imai T, Rutten SJ, Fairweather JK, Pelosi L, Bulone V, Driguez H, Fincher GB: Mutated barley (1,3)-b-D-glucan

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endohydrolases synthesize crystalline (1,3)-b-D-glucans. J Biol Chem 2002, 277:30102-30111. 84. Fairweather JK, Hrmova M, Rutten SJ, Fincher GB, Driguez H: Synthesis of complex oligosaccharides by using a mutated (1,3)-b-D-glucan endohydrolase from barley. Chemistry 2003, 9:2603-2610. 85. Jahn M, Stoll D, Warren RA, Szabo L, Singh P, Gilbert HJ, Ducros VM, Davies GJ, Withers SG: Expansion of the glycosynthase repertoire to produce defined mannooligosaccharides. Chem Commun (Camb) 2003: 1327-1329. 86. Okuyama M, Mori H, Watanabe K, Kimura A, Chiba S: a-Glucosidase mutant catalyzes ‘a-glycosynthase’-type reaction. Biosci Biotechnol Biochem 2002, 66:928-933. 87. Aharoni A, Thieme K, Chiu CP, Buchini S, Lairson LL,  Chen H, Strynadka NC, Wakarchuk WW, Withers SG: High-throughput screening methodology for the directed evolution of glycosyltransferases. Nat Methods 2006, 3:609-614. By use of reversibly transported fluorescent lactose derivatives as acceptors and an E. Coli cell line in which b-galactosidase and aldolase were deleted, an E. Coli cell containing plasmid encoded CMP-synthase and an active sialyltransferase is rendered fluorescent and, thus, sortable via product entrapment.

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