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We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP–glucoses and UDP–sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

A two-step strategy for disaccharide modulation using vancomycin as a model is reported. The strategy relies upon a glycosyltransferase-catalyzed 'reverse' reaction to enable the facile attachment of an alkoxyamine-bearing sugar to the vancomycin core. Neoglycosylation of the corresponding aglycon led to a novel set of vancomycin 1,6-disaccharide variants. While the in vitro antibacterial properties of corresponding vancomycin 1,6-disaccharide analogs were equipotent to the parent antibiotic, the chemoenzymatic method presented is expected to be broadly applicable.

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution ( Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min(–1) on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min(–1). UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.

Glycosyltransferases (GTs) constitute a predominant enzyme superfamily responsible for the attachment of carbohydrate moieties to a wide array of acceptors that include nucleic acids, polysaccharides, proteins, lipids, carbohydrates and medicinally relevant secondary metabolites (1, 2). The majority of GTs are LeLoir (sugar nucleotide-dependent) enzymes and utilize nucleoside diphosphate sugars (NDP-sugars) as donors for glycosidic bond formation (Fig. 1a). Recent studies have revealed certain GT-catalyzed reactions from bacterial secondary metabolism to be reversible, presenting new GT-catalyzed methods for NDP-sugar synthesis as well as the GT-catalyzed exchange (Fig. 1b) or transfer (Fig. 1c) of sugars attached to both simple glycoside sugar donors(3, 4) and complex natural product glycosides including glycopeptides, enediynes(5), macrolides(6), macrolactams(7), (iso)flavonoids(8) and polyenes(9). Of the few examples which employed ‘activated’ sugar donors (e.g., glycosyl halides or aromatic glycosides) for GT-catalyzed transglycosylation(3, 4), only indirect evidence for the intermediacy of sugar nucleotides was provided. Among GT-catalyzed ‘reverse’ reactions where NDP-sugar formation has been confirmed, NDP-sugar formation was thermodynamically disfavored (i.e., with NDP-sugar as product, Keq < 1)(5, 10, 11) and typically required a 10- to 100-fold excess of NDP for sugar nucleotide production. In an effort to address the severe thermodynamic limitations of GT-catalyzed reactions run in reverse, herein we report the use of specific activated aromatic glycosides as donors in such reactions dramatically alters the equilibrium of GT-catalyzed reactions and thereby enables a variety of novel transformations (Fig. 1d) including: i) an unique platform for the efficient enzymatic syntheses of novel NDP-sugars, ii) a coupled GT-catalyzed platform for the differential glycosylation of small molecules (including natural products and synthetic drugs/targets), and iii) a colorimetric readout upon glycosyltransfer amenable to high throughput formats for glycodiversification and glycoengineering which can be coupled to nearly any downstream sugar nucleotide-utilizing enzyme. Fig. 1 Representative GT-catalyzed reactions. a) Classical GT-catalyzed transformation wherein the sugar, presented in the form of a sugar nucleotide donor, is conjugated to an acceptor target of interest to provide a thermodynamically-favored glycoside product. ...