[This corrects the article DOI: 10.1371/journal.pone.0161209.].

Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an α-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mutations inferred by molecular docking simulations were generated and screened for quercetin glucosylation using sucrose as a glucosyl donor. Only a limited number of variants (22) were retained on the basis of quercetin conversion and product profile. Their acceptor promiscuity towards five other flavonoids was subsequently assessed, and the automated screening effort revealed variants showing remarkable ability for luteolin, morin and naringenin glucosylation with conversion ranging from 30% to 90%. Notably, naringenin and morin, a priori considered as recalcitrant compounds to glucosylation using this α-transglucosylases, could also be modified. The approach reveals the potential of small platforms of engineered GH70 α-transglucosylases and opens up the diversity of flavonoid glucosides to molecular structures inaccessible yet.

2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l −1 DHB with a molar yield of 0.15.

The α-(1→2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1→2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes.

Flavonoids constitute an important class of bioactive molecules, the physicochemical properties of which can be modulated by glucosylation. A structurally guided approach has been used to isolate glucansucrases modified in their acceptor-binding site and specialized for luteolin glucosylation. Of a small-size library, we isolate mutants showing up to an 8-fold increase in flavonoid conversion rate over that observed with the parental enzyme. Di- and triglucosylated luteolin derivatives never described before have been obtained. They exhibit 282- and 17708-fold increases in water solubility, respectively, and are protected from oxidation by the glucosylation reaction. Molecular docking enables insight into the product specificity of the best mutants. These results demonstrate that atransglucosylase engineering is a powerful means to generate highly specific catalysts for flavonoid glucosylation and deliver new structural scaffolds with increased bioavailability and high relevance for therapeutic applications.

Experimental design and Response Surface Methodology (RSM) were used to optimize the production of ∆N123-GBD-CD2, an α-(1 → 2) branching sucrase previously reported as mainly produced in inclusion bodies. The ∆N123-GBD-CD2 encoding gene was cloned into two expression vectors in fusion with 6xHis tag or Strep tag II encoding sequences at 5′ and 3′ ends of the gene and expressed in five Escherichia coli strains. Three host-vector combinations were first selected on the basis of the amount of soluble enzyme produced. RSM with Box-Behnken design was used to optimize the expression conditions in an auto-inducible medium. Five factors were considered, i.e. culture duration, temperature and the concentrations of glycerol, lactose inducer and glucose repressor. The design consisted of three blocks of 45 assays performed in deep well microplates. The regression models were built and fitted well to the experimental data (R 2 coefficient >94 %). The best response (production level of soluble enzyme) was obtained with E. coli BL21 Star DE3 cells transformed with the pET-55 vector. Using the predicted optimal conditions, 5,740 U L−1 of culture of soluble enzyme was produced in microtiter plates and more than 12,000 U L−1 of culture in Erlenmeyer flask, which represents a 165-fold increase compared to the production levels previously reported.

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.