Glucodiversification des flavonoïdes par ingénierie d’enzymes actives sur saccharose

Flavonoid glycosides are natural plant secondary metabolites exhibiting many physicochemical and biological properties. Glycosylation usually improves flavonoid solubility but access to flavonoid glycosides is limited by their low production levels in plants. In this thesis work, the focus was placed on the development of new glucodiversification routes of natural flavonoids by taking advantage of protein engineering. Two biochemically and structurally characterized recombinant transglucosylases, the amylosucrase from Neisseria polysaccharea and the α-(1→2) branching sucrase, a truncated form of the dextransucrase from L. Mesenteroides NRRL B-1299, were selected to attempt glucosylation of different flavonoids, synthesize new α-glucoside derivatives with original patterns of glucosylation and hopefully improved their water-solubility. First, a small-size library of amylosucrase variants showing mutations in their acceptor binding site was screened in the presence of sucrose (glucosyl donor) and luteolin acceptor. A screening procedure was developed. It allowed isolating several mutants improved for luteolin glucosylation and synthesizing of novel luteolin glucosides, which exhibited up to a 17,000-fold increase of solubility in water. To attempt glucosylation of other types of flavonoids, the α-(1→2) branching sucrase, naturally designed for acceptor reaction, was preferred. Experimental design and Response Surface Methodology were first used to optimize the production of soluble enzyme and avoid inclusion body formation. Five parameters were included in the design: culture duration, temperature and concentrations of glycerol, lactose inducer and glucose repressor. Using the predicted optimal conditions, 5740 U. L-1of culture of soluble enzyme were obtained in microtiter plates, while no activity was obtained in the soluble fraction when using the previously reported method of production. A structurally-guided approach, based on flavonoids monoglucosides docking in the enzyme active site, was then applied to identify mutagenesis targets and generate libraries of several thousand variants. They were screened using a rapid pH-based screening assay, implemented for this purpose. This allowed sorting out mutants still active on sucrose that were subsequently assayed for both quercetin and diosmetin glucosylation. A small set of 23 variants, constituting a platform of enzymes improved for the glucosylation of these two flavonoids was retained and evaluated for the glucosylation of a six distinct flavonoids. Remarkably, the promiscuity generated in this platform allowed isolating several variants much more efficient than the wild-type enzyme. They produced different glucosylation patterns, and provided valuable information to further design and improve flavonoid glucosylation enzymatic tools.Les flavonoïdes glycosylés sont des métabolites secondaires d’origine végétale, qui présentent de nombreuses propriétés physico-chimiques et biologiques intéressantes pour des applications industrielles. La glycosylation accroît généralement la solubilité de ces flavonoïdes mais leurs faibles niveaux de production dans les plantes limitent leur disponibilité. Ces travaux de thèse portent donc sur le développement de nouvelles voies de gluco-diversification des flavonoïdes naturels, en mettant à profit l’ingénierie des protéines. Deux transglucosylases recombinantes, structurellement et biochimiquement caractérisées, l'amylosaccharase de Neisseria polysaccharea et la glucane-saccharase de branchement α-(1→2), forme tronquée de la dextran-saccharase de L. Mesenteroides NRRL B-1299, ont été sélectionnées pour la biosynthèse de nouveaux flavonoïdes, possédant des motifs originaux d’α-glycosylation, et potentiellement une solubilité accrue dans l'eau. Dans un premier temps, une librairie de petite taille de mutants de l’amylosaccharase, ciblée sur le site de liaison à l’accepteur, à été criblée en présence de saccharose (donneur d’unité glycosyl) et de lutéoline comme accepteur. Une méthode de screening a donc été développée, et a permis d’isoler des mutants améliorés pour la synthèse de nouveaux glucosides de lutéoline, jusqu’à 17000 fois plus soluble dans l’eau que la lutéoline aglycon. Afin de glucosyler d’autres flavonoïdes, la glucane-saccharase de branchement α-(1→2), a été préférentiellement sélectionnée. Des plans expérimentaux alliés à une méthodologie en surface de réponse ont été réalisés pour optimiser la production de l’enzyme sous forme soluble et éviter la formation de corps d’inclusion. Cinq paramètres ont été ainsi analysés : le temps de culture, la température, et les concentrations en glycérol, lactose (inducteur) et glucose (répresseur). En appliquant les conditions optimales prédites, 5740 U.L-1 de culture d’enzyme soluble ont été produites en microplaques, alors qu’aucune activité n’était retrouvée dans la fraction soluble, lors de l’utilisation de la méthode de production précédemment utilisée. Finalement, Une approche de modélisation moléculaire, structurellement guidés par l’arrimage de flavonoïdes monoglucosylés dans le site actif de l’enzyme, a permis d’identifier des cibles de mutagenèse et de générer des libraries de quelques milliers de variants. Une méthode rapide de criblage sur milieu solide, basée sur la visualisation colorimétrique d’un changement de pH, a été mise au point. Les mutants encore actifs sur saccharose ont été sélectionnés puis analysés sur leur capacités à glucosyler la quercétine et la diosmétine. Une petite série de 23 mutants a ainsi été retenue comme plate-forme d’enzymes améliorées dédiées à la glucosylation de flavonoïdes et a été évalués pour la glycosylation de six flavonoïdes distincts. La promiscuité, remarquablement générée dans cette plateforme, à permis d’isoler quelques mutants beaucoup plus efficaces que l’enzyme sauvage, produisant des motifs de glucosylation différents et fournissant des informations intéressante pour le design et l’amélioration des outils enzymatiques de glucosylation des flavonoïdes

Optimizing the production of an α-(1→2) branching sucrase in [i]Escherichia coli[/i] using statistical design

Experimental design and Response Surface Methodology (RSM) were used to optimize the production of a dagger N-123-GBD-CD2, an alpha-(1 -> aEuro parts per thousand 2) branching sucrase previously reported as mainly produced in inclusion bodies. The a dagger N-123-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

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70

International audienceThe alpha-(1 -> 2) branching sucrase Delta N-123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer of D-glucosyl units from sucrose to dextrans or gluco-oligosaccharides via the formation of alpha-(1 -> 2) glucosidic linkages. The first structures of Delta N-123-GBD-CD2 in complex with D-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 alpha-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes

Engineering a branching sucrase for flavonoid glucoside diversification

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 alpha-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 alpha-transglucosylases, could also be modified. The approach reveals the potential of small platforms of engineered GH70 alpha-transglucosylases and opens up the diversity of flavonoid glucosides to molecular structures inaccessible yet

Correction: A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

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

A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-β 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies

Extending the Structural Diversity of alpha-Flavonoid Glycosides with Engineered Glucansucrases

International audienceFlavonoids 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

¹H NMR of MAβ_1–40 and MAβ_1–42 compared with Aβ_1–40.

<p>Samples of MAβ in water are diluted to 100 μM in 100 mM phosphate buffer pH 7 in D₂O. Synthetic Aβ_1–40 is dissolved in NaOD and diluted to 100 μM under the same conditions. Signals at 4.75 ppm correspond to H₂O, and signals at 3.25 ppm and 1.25 ppm correspond to traces of ethanol.</p

Purification protocol of MAβ_1–42 IBs dissolved in 8M urea.

<p>Lane 1: 10–250 kDa protein marker, lane 2: IBs after urea denaturation, lane 3: after passing through DEAE resin, lane 4: after 30-kDa ultrafiltration, lane 5: after 3-kDa concentration.</p

Purification monitoring of MAβ.

<p><b>A.</b> Simplified scheme of the purification procedure. Circled letters correspond to samples collected for either UV/Vis measurements or gel analysis. Supernatant S1 and S2, and pellet P1 and P2 correspond to the supernatant and the precipitate obtained for the two cycles of denaturation/neutralisation. <b>B.</b> SDS-PAGE at different steps of purification, compared with 10–250 kDa protein marker (lane M). <b>C.</b> UV-Vis spectra at pH 12 after IBs denaturation (D), after denaturation/neutralisation cycles (S1+S2), after 30-kDa ultrafiltration (F), after concentration (C), and after size exclusion chromatography (SEC).</p