Leloir Glycosyltransferases Research Articles

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation.

Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5'-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix (McSuSy), a single-cell green alga, was overexpressed in Escherichia coli, and its enzymatic properties were characterized. In the direction of sucrose cleavage, the specific activity of the recombinant McSuSy is 9.39 U/mg at 37°C and pH 7.0, and the optimum temperature and pH were 60°C and pH 7.0, respectively. Its nucleotide preference for uridine 5'-diphosphate (UDP) was similar to plant SuSys, and the enzyme activity remained relatively high when the DMSO concentration below 25%. The mutation of the predicted N-terminal phosphorylation site (S31D) significantly stimulated the activity of McSuSy. When the mutant S31D of McSuSy was applied by coupling the engineered Stevia glycosyltransferase UGT76G1 in a one-pot two-enzyme reaction at 10% DMSO, 50 g/L rebaudioside E was transformed into 51.06 g/L rebaudioside M in 57 h by means of batch feeding, with a yield of 76.48%. This work may reveal the lower eukaryotes as a promising resource for SuSys of industrial interest.

Whole-cell bioconversion using non-Leloir transglycosylation reactions: a review.

Microbial biocatalysts are evolving technological tools for glycosylation research in food, feed and pharmaceuticals. Advances in bioengineered Leloir and non-Leloir carbohydrate-active enzymes allow for whole-cell biocatalysts to curtail production costs of purified enzymes while enhancing glucan synthesis through continued enzyme expression. Unlike sugar nucleotide-dependent Leloir glycosyltransferases, non-Leloir enzymes require inexpensive sugar donors and can be designed to match the high value, yield and selectivity of the former. This review addresses the current state of bacterial cell-based production of glucans and glycoconjugates via transglycosylation, and describes how alterations made to microbial hosts to surpass purified enzymes as the preferred mode of catalysis are steadily being acquired through genetic engineering, rational design and process optimization. A comprehensive exploration of relevant literature has been summarized to describe whole-cell biocatalysis in non-Leloir glycosylation reactions with various donors and acceptors, and the characterization, application and latest developments in the optimization of their use.

Evaluation of double expression system for co-expression and co-immobilization of flavonoid glucosylation cascade

Glucosylation cascade consisting of Leloir glycosyltransferase and sucrose synthase with in situ regeneration system of expensive and low available nucleotide sugars is a game-changing strategy for enzyme-based production of glycoconjugates of relevant natural products. We designed a stepwise approach including co-expression and one-step purification and co-immobilization on glass-based EziG resins of sucrose synthase from Glycine max (GmSuSy) with promiscuous glucosyltransferase YjiC from Bacillus licheniformis to produce efficient, robust, and versatile biocatalyst suited for preparative scale flavonoid glucosylation. The undertaken investigations identified optimal reaction conditions (30 °C, pH 7.5, and 10 mM Mg2+) and the best-suited carrier (EziG Opal). The prepared catalyst exhibited excellent reusability, retaining up to 96% of initial activity after 12 cycles of reactions. The semi-preparative glucosylation of poorly soluble isoflavone Biochanin A resulted in the production of 73 mg Sissotrin (Biochanin A 7-O-glucoside). Additionally, the evaluation of the designed double-controlled, monocistronic expression system with two independently induced promoters (rhaBAD and trc) brought beneficial information for dual-expression plasmid design.Key points• Simultaneous and titratable expression from two independent promoters is possible, although full control over the expression is limited.• Designed catalyst managed to glucosylate poorly soluble isoflavone.• The STY of Sissotrin using the designed catalyst reached 0.26 g/L∙h∙g of the resin.Graphical

Nano-fibrillated cellulose-based scaffolds for enzyme (co)-immobilization: Application to natural product glycosylation by Leloir glycosyltransferases

Polysaccharide-based scaffolds are promising carriers for enzyme immobilization. Here, we demonstrate a porous scaffold prepared by direct-ink-writing 3D printing of an ink consisting of nanofibrillated cellulose, carboxymethyl cellulose and citric acid for immobilization application. Negative surface charge introduced by the components made the scaffold amenable for an affinity-like immobilization via the cationic protein module Zbasic2. Zbasic2 fusions of two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z-CGT; sucrose synthase, Z-SuSy) were immobilized individually, or co-immobilized, and applied to synthesize the natural C-glycoside nothofagin. The cascade reaction involved β-C-glycosylation of phloretin (10 mM, ~90 % conversion) from UDP-glucose, provided from sucrose and catalytic amounts of UDP (1.0 mM). Enzymes were co-immobilized at ~65 mg protein/g carrier to receive activities of 9.5 U/g (Z-CGT) and 4.5 U/g (Z-SuSy) in 22–33 % yield (protein) and an effectiveness of 23 % (Z-CGT) and 13 % (Z-SuSy). The scaffold-bound enzymes were recyclable for 5 consecutive reactions.

Synthesis of Potential Glycosyl Transferase Inhibitors by Thio‐Click Reactions

AbstractLeloir glycosyltransferases play an important role in many fundamental processes of living systems by catalyzing biological glycosylations. Neutral analogues of nucleotide sugars, as potential donor‐substrate inhibitors of Leloir glycosyltransferases, are of prime interest in medicinal chemistry. We report the development of S‐linked sugar‐nucleotide analogues by replacing the α‐O‐pyrophosphate bridge with an electroneutral ethyl or propyl linker by radical mediated hydrothiolation of 2‐substituted glycals and olefinic nucleoside analogues. UDP‐glucose and UDP‐GlcNAc analogues were assembled from 2‐substituted glycals, 1,2‐ethanedithiols and uridine 4’‐exomethylene derivative by two subsequent thio‐click reactions. Morpholino type mimetics of UDP‐, GDP‐ and CMP‐sugars were constructed by photoinitiated additions of 1‐thiosugars (Glc, GlcNAc, Gal, Man) and 2‐thio‐Neu5Ac onto N‐allyl morpholinos.

Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C-glycoside nothofagin.

C‐glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Z basic2, were co‐immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous production of the natural C‐glycoside nothofagin. The overall reaction was 3ʹ‐C‐β‐glycosylation of the polyphenol phloretin from uridine 5ʹ‐diphosphate (UDP)‐glucose that was released in situ from sucrose and UDP. Using solid catalyst optimized for total (∼28 mg/g) as well as relative protein loading (CGT/SuSy = ∼1) and assembled into a packed bed (1 ml), we demonstrate flow synthesis of nothofagin (up to 52 mg/ml; 120 mM) from phloretin (≥95% conversion) solubilized by inclusion complexation in hydroxypropyl β‐cyclodextrin. About 1.8 g nothofagin (90 ml; 12–26 mg/ml) were produced continuously over 90 reactor cycles (2.3 h/cycle) with a space‐time yield of approximately 11 mg/(ml h) and a total enzyme turnover number of up to 2.9 × 103 mg/mg (=3.8 × 105 mol/mol). The co‐immobilized enzymes exhibited useful effectiveness (∼40% of the enzymes in solution), with limitations on the conversion rate arising partly from external liquid–solid mass transfer of UDP under packed‐bed flow conditions. The operational half‐life of the catalyst (∼200 h; 30°C) was governed by the binding stability of the glycosyltransferases (≤35% loss of activity) on the solid carrier. Collectively, the current study shows integrated process technology for flow synthesis with co‐immobilized sugar nucleotide‐dependent glycosyltransferases, using efficient glycosylation from sucrose via the internally recycled UDP‐glucose. This provides a basis from engineering science to promote glycosyltransferase applications for natural product glycosides and oligosaccharides.

Selective β-Mono-Glycosylation of a C15-Hydroxylated Metabolite of the Agricultural Herbicide Cinmethylin Using Leloir Glycosyltransferases.

Cinmethylin is a well-known benzyl-ether derivative of the natural terpene 1,4-cineole that is used industrially as a pre-emergence herbicide in grass weed control for crop protection. Cinmethylin detoxification in plants has not been reported, but in animals, it prominently involves hydroxylation at the benzylic C15 methyl group. Here, we show enzymatic β-glycosylation of synthetic 15-hydroxy-cinmethylin to prepare a putative phase II detoxification metabolite of the cinmethylin in plants. We examined eight Leloir glycosyltransferases for reactivity with 15-hydroxy cinmethylin and revealed the selective formation of 15-hydroxy cinmethylin β-d-glucoside from uridine 5′-diphosphate (UDP)-glucose by the UGT71E5 from safflower (Carthamus tinctorius). The UGT71E5 showed a specific activity of 431 mU/mg, about 300-fold higher than that of apple (Malus domestica) UGT71A15 that also performed the desired 15-hydroxy cinmethylin mono-glycosylation. Bacterial glycosyltransferases (OleD from Streptomyces antibioticus, 2.9 mU/mg; GT1 from Bacillus cereus, 60 mU/mg) produced mixtures of 15-hydroxy cinmethylin mono- and disaccharide glycosides. Using UDP-glucose recycling with sucrose synthase, 15-hydroxy cinmethylin conversion with UGT71E5 efficiently provided the β-mono-glucoside (≥95% yield; ∼9 mM) suitable for biological studies.

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogs at the donor site (-1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the second-most reactive gauche,trans (gt) conformation, and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help progress the design and development of improved, conformationally-restricted GT inhibitors that take advantage of these inherent side chain preferences.

Phylogenomic Analyses of Nucleotide-Sugar Biosynthetic and Interconverting Enzymes Illuminate Cell Wall Composition in Fungi.

The fungi are an enormously successful eukaryotic lineage that has colonized every aerobic habitat on Earth. This spectacular expansion is reflected in the dynamism and diversity of the fungal cell wall, a matrix of polysaccharides and glycoproteins pivotal to fungal life history strategies and a major target in the development of antifungal compounds. Cell wall polysaccharides are typically synthesized by Leloir glycosyltransferases, enzymes that are notoriously difficult to characterize, but their nucleotide-sugar substrates are well known and provide the opportunity to inspect the monosaccharides available for incorporation into cell wall polysaccharides and glycoproteins. In this work, we have used phylogenomic analyses of the enzymatic pathways that synthesize and interconvert nucleotide-sugars to predict potential cell wall monosaccharide composition across 491 fungal taxa. The results show a complex evolutionary history of these cell wall enzyme pathways and, by association, of the fungal cell wall. In particular, we see a significant reduction in monosaccharide diversity during fungal evolution, most notably in the colonization of terrestrial habitats. However, monosaccharide distribution is also shown to be varied across later-diverging fungal lineages.IMPORTANCE This study provides new insights into the complex evolutionary history of the fungal cell wall. We analyzed fungal enzymes that convert sugars acquired from the environment into the diverse sugars that make up the fundamental building blocks of the cell wall. Species-specific profiles of these nucleotide-sugar interconverting (NSI) enzymes for 491 fungi demonstrated multiple losses and gains of NSI proteins, revealing the rich diversity of cell wall architecture across the kingdom. Pragmatically, because cell walls are essential to fungi, our observations of variation in sugar diversity have important implications for the development of antifungal compounds that target the sugar profiles of specific pathogens.

Multi‐enzyme Cascades for the In Vitro Synthesis of Guanosine Diphosphate L‐Fucose

AbstractRecombinant Leloir glycosyltransferases can be exploited to synthesize a wide range of HMOs using in vitro biocatalytic reactions. However, high costs and unavailability of bulk amounts of most nucleotide sugars, such as guanosine diphosphate L‐fucose (GDP‐Fuc), are major obstacles for the efficient large‐scale synthesis. Here, we report two novel multi‐enzyme cascades for the synthesis of GDP‐Fuc from readily available and low cost precursors. The first cascade was developed to produce GDP‐Fuc from guanosine (Guo), fucose (Fuc), polyphosphate (PolyPn) and catalytic amounts of adenine triphosphate (ATP). GDP‐Fuc was produced with a final concentration of 7 mM (4.1 g/L) and a reaction yield of 68 % from Guo and Fuc within 48 h with a biocatalyst load of 0.34 genzyme/gproduct. A second cascade, consisting of ten enzymes and eleven reactions was developed to carry out the synthesis from mannose (Man), Guo, PolyPn, L‐glutamine (L‐Glu) and catalytic amounts of ATP, and nicotinamide adenine dinucleotide phosphate (NADPH). Utilizing this cascade, GDP‐Fuc was produced with a final concentration of 7.6 mM (4.5 g/L) and a reaction yield of 72 % in a reaction time of 48 h with a biocatalyst load of 0.97 genzyme/gproduct. Finally, a method for chromatographic purification of GDP‐Fuc was established achieving product purities of 90.5 %.

Anomeric Selectivity of Trehalose Transferase with Rare l-Sugars.

Retaining LeLoir glycosyltransferases catalyze the formation of glycosidic bonds between nucleotide sugar donors and carbohydrate acceptors. The anomeric selectivity of trehalose transferase from Thermoproteus uzoniensis was investigated for both d- and l-glycopyranose acceptors. The enzyme couples a wide range of carbohydrates, yielding trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for d-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for l-glycopyranose acceptors, while (S)-selectivity was retained for both types of sugar acceptors. Comparison of protein crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.

Efficient enzyme formulation promotes Leloir glycosyltransferases for glycoside synthesis

Sugar nucleotide-dependent (Leloir) glycosyltransferases are powerful catalysts for glycoside synthesis. Their applicability can be limited due to elaborate production of enzyme preparations deployable in biocatalytic processes. Here, we show that efficient enzyme formulation promotes glycosyltransferases for the synthesis of the natural C-glycoside nothofagin. Adding Brij-35 detergent (1 %, w/v) during sonication of the E. coli BL21-Gold (DE3) expression strain, recovery of Oryza sativa C-glycosyltransferase was enhanced by ∼3-fold, partly due to the release of enzyme activity trapped in insoluble pellet. Freeze drying of the resulting cell-free extract (∼17 U ml−1) reduced the volume ∼20-fold and gave ∼55 mg solids ml−1 liquid processed, with 83 % retention of the original activity and a specific activity of 0.20 U mg−1 solids. The Glycine max sucrose synthase was processed analogously, giving a solid enzyme preparation of 0.28 U mg-1 in 63 % yield. Both enzyme formulations were stable for several weeks. The glycosyltransferase cascade reaction for 3′-β-C-glucosylation of phloretin (60 mM; as inclusion complex with hydroxypropyl-β-cyclodextrin) from UDP-glucose (generated in situ by sucrose synthase from 500 mM sucrose and 0.5 mM UDP) showed excellent performance metrics (≥ 98 % yield; 3.2 g l−1 h−1 space-time yield; ∼90 regeneration cycles for UDP). Collectively, our study demonstrates a facile procedure for solid glycosyltransferase formulations practically usable in glycoside synthesis.

Leloir glycosyltransferases of natural product C-glycosylation: structure, mechanism and specificity.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.

Enzymatic Synthesis of Glycans and Glycoconjugates.

Glycoconjugates have great potential to improve human health in a multitude of different ways and fields. Prominent examples are human milk oligosaccharides and glycosaminoglycans. The typical choice for the production of homogeneous glycoconjugates is enzymatic synthesis. Through the availability of expression and purification protocols, recombinant Leloir glycosyltransferases are widely applied as catalysts for the synthesis of a wide range of glycoconjugates. Extensive utilization of these enzymes also depends on the availability of activated sugars as building blocks. Multi-enzyme cascades have proven a versatile technique to synthesize and in situ regenerate nucleotide sugar.In this chapter, the functions and mechanisms of Leloir glycosyltransferases are revisited, and the advantage of prokaryotic sources and production systems is discussed. Moreover, in vivo and in vitro pathways for the synthesis of nucleotide sugar are reviewed. In the second part, recent and prominent examples of the application of Leloir glycosyltransferase are given, i.e., the synthesis of glycosaminoglycans, glycoconjugate vaccines, and human milk oligosaccharides as well as the re-glycosylation of biopharmaceuticals, and the status of automated glycan assembly is revisited.

WITHDRAWN: Efficient enzyme formulation promotes Leloir glycosyltransferases for glycoside synthesis

The Publisher regrets that this article is an accidental duplication of an article that has already been published in BIOTEC, 322C (2020) 74-78, https://doi.org/10.1016/j.jbiotec.2020.06.023. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach.

Enzymes are nature’s catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.

Two Trifunctional Leloir Glycosyltransferases as Biocatalysts for Natural Products Glycodiversification.

Two promiscuous Bacillus licheniformis glycosyltransferases, YdhE and YojK, exhibited prominent stereospecific but nonregiospecific glycosylation activity of 20 different classes of 59 structurally different natural and non-natural products. Both enzymes transferred various sugars at three nucleophilic groups (OH, NH2, SH) of diverse compounds to produce O-, N-, and S-glycosides. The enzymes also displayed a catalytic reversibility potential for a one-pot transglycosylation, thus bestowing a cost-effective application in biosynthesis of glycodiversified natural products in drug discovery.

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

AbstractImmobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space‐time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost‐benefit analysis prior to scale‐up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non‐sulfated human natural killer cell‐1 (HNK‐1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5′‐ diphospho‐α‐ d‐galactose (UDP‐Gal) and uridine 5′‐diphospho‐α‐d‐glucuronic acid (UDP‐GlcA), the donor substrates for the Leloir glycosyltransferases β4‐galactosyltransferase and β3‐glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS‐system connected to the microreactor. The high synthesis yield of 96% for the non‐sulfated HNK‐1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system.magnified image

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability.

LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1→1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, Keq, for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control.IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.