BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project

Tailored enzymes for programmed chemo-enzymatic synthesis of antigenic oligosaccharides

Les travaux de recherche présentés dans cette thèse visent à explorer des stratégies multidisciplinaires et synergiques afin de développer des voies chimio-enzymatiques «programmées» qui tirent parti de la promiscuité de substrat innée et de l’ingénierie assistée par ordinateur des α-transglucosylases. Le but est ainsi de produire des glycobriques, facilement assemblables en molécules biologiquement actives et mimant le fragment polysaccharidique antigénique - O-antigène (O-Ag) - des lipopolysaccharides de Shigella flexneri. Une version protégée (ABC’D’) a tout d’abord été synthétisée par nos collaborateurs de l’Institut Pasteur. Nous avons ensuite évalué le potentiel des sucrases de branchement (BRS) de la famille 70 des Glycoside Hydrolases pour la glucosylation de ABC’D’. Ces enzymes présentent une grande promiscuité vis-à-vis des substrats accepteurs. Au final, nos résultats démontrent la polyvalence des BRS et de leurs mutants pour accéder à une large gamme de profils de glucosylation à partir d’une molécule accepteur commune, ABC’D’. Ces travaux ouvrent également de nouvelles voies pour améliorer les performances catalytiques de ces mutants et pour mieux comprendre les déterminants moléculaires et dynamiques qui pourraient jouer un rôle dans la reconnaissance des accepteurs et leur glucosylation sélective, ce qui pourra offrir de nouvelles possibilités pour la synthèse des haptènes de S. flexneri.The research work presented in this thesis aimed at exploring multidisciplinary and synergistic strategies to develop “programmed” chemo-enzymatic pathways that take advantage of innate substrate promiscuity and computer-aided engineering of α-transglucosylases to produce glycobricks, easy-to-assemble into biologically active molecules mimicking the antigenic polysaccharide moiety – O-antigen (O-Ag) – of the Shigella flexneri lipopolysaccharides. A lightly protected ABC’D’ was first chemically synthesized by our collaborators from the Pasteur Institute. Next, we evaluated the potential of branching sucrases (BRS) from the Glycoside Hydrolase family 70 to glucosylate ABC’D’. They present the advantage of displaying a large promiscuity toward acceptor substrates. Overall, our results demonstrate the versatility of branching sucrases and their mutants to access a broad range of glucosylation patterns from a common tetrasaccharide acceptor molecule, ABC’D’. It also opens new paths for improving catalytic performances of these mutants and better understanding molecular and dynamical determinants that could play a role in acceptor recognition and its site-selective glucosylation that will hopefully offer new opportunities for the synthesis of S. flexneri haptens

Conception « à façon » d'enzymes pour la synthèse chimio-enzymatique programmée d'oligosaccharides antigéniques

The research work presented in this thesis aimed at exploring multidisciplinary and synergistic strategies to develop “programmed” chemo-enzymatic pathways that take advantage of innate substrate promiscuity and computer-aided engineering of α-transglucosylases to produce glycobricks, easy-to-assemble into biologically active molecules mimicking the antigenic polysaccharide moiety – O-antigen (O-Ag) – of the Shigella flexneri lipopolysaccharides. A lightly protected ABC’D’ was first chemically synthesized by our collaborators from the Pasteur Institute. Next, we evaluated the potential of branching sucrases (BRS) from the Glycoside Hydrolase family 70 to glucosylate ABC’D’. They present the advantage of displaying a large promiscuity toward acceptor substrates. Overall, our results demonstrate the versatility of branching sucrases and their mutants to access a broad range of glucosylation patterns from a common tetrasaccharide acceptor molecule, ABC’D’. It also opens new paths for improving catalytic performances of these mutants and better understanding molecular and dynamical determinants that could play a role in acceptor recognition and its site-selective glucosylation that will hopefully offer new opportunities for the synthesis of S. flexneri haptens.Les travaux de recherche présentés dans cette thèse visent à explorer des stratégies multidisciplinaires et synergiques afin de développer des voies chimio-enzymatiques «programmées» qui tirent parti de la promiscuité de substrat innée et de l’ingénierie assistée par ordinateur des α-transglucosylases. Le but est ainsi de produire des glycobriques, facilement assemblables en molécules biologiquement actives et mimant le fragment polysaccharidique antigénique - O-antigène (O-Ag) - des lipopolysaccharides de Shigella flexneri. Une version protégée (ABC’D’) a tout d’abord été synthétisée par nos collaborateurs de l’Institut Pasteur. Nous avons ensuite évalué le potentiel des sucrases de branchement (BRS) de la famille 70 des Glycoside Hydrolases pour la glucosylation de ABC’D’. Ces enzymes présentent une grande promiscuité vis-à-vis des substrats accepteurs. Au final, nos résultats démontrent la polyvalence des BRS et de leurs mutants pour accéder à une large gamme de profils de glucosylation à partir d’une molécule accepteur commune, ABC’D’. Ces travaux ouvrent également de nouvelles voies pour améliorer les performances catalytiques de ces mutants et pour mieux comprendre les déterminants moléculaires et dynamiques qui pourraient jouer un rôle dans la reconnaissance des accepteurs et leur glucosylation sélective, ce qui pourra offrir de nouvelles possibilités pour la synthèse des haptènes de S. flexneri

Conception « à façon » d'enzymes pour la synthèse chimio-enzymatique programmée d'oligosaccharides antigéniques

The research work presented in this thesis aimed at exploring multidisciplinary and synergistic strategies to develop “programmed” chemo-enzymatic pathways that take advantage of innate substrate promiscuity and computer-aided engineering of α-transglucosylases to produce glycobricks, easy-to-assemble into biologically active molecules mimicking the antigenic polysaccharide moiety – O-antigen (O-Ag) – of the Shigella flexneri lipopolysaccharides. A lightly protected ABC’D’ was first chemically synthesized by our collaborators from the Pasteur Institute. Next, we evaluated the potential of branching sucrases (BRS) from the Glycoside Hydrolase family 70 to glucosylate ABC’D’. They present the advantage of displaying a large promiscuity toward acceptor substrates. Overall, our results demonstrate the versatility of branching sucrases and their mutants to access a broad range of glucosylation patterns from a common tetrasaccharide acceptor molecule, ABC’D’. It also opens new paths for improving catalytic performances of these mutants and better understanding molecular and dynamical determinants that could play a role in acceptor recognition and its site-selective glucosylation that will hopefully offer new opportunities for the synthesis of S. flexneri haptens.Les travaux de recherche présentés dans cette thèse visent à explorer des stratégies multidisciplinaires et synergiques afin de développer des voies chimio-enzymatiques «programmées» qui tirent parti de la promiscuité de substrat innée et de l’ingénierie assistée par ordinateur des α-transglucosylases. Le but est ainsi de produire des glycobriques, facilement assemblables en molécules biologiquement actives et mimant le fragment polysaccharidique antigénique - O-antigène (O-Ag) - des lipopolysaccharides de Shigella flexneri. Une version protégée (ABC’D’) a tout d’abord été synthétisée par nos collaborateurs de l’Institut Pasteur. Nous avons ensuite évalué le potentiel des sucrases de branchement (BRS) de la famille 70 des Glycoside Hydrolases pour la glucosylation de ABC’D’. Ces enzymes présentent une grande promiscuité vis-à-vis des substrats accepteurs. Au final, nos résultats démontrent la polyvalence des BRS et de leurs mutants pour accéder à une large gamme de profils de glucosylation à partir d’une molécule accepteur commune, ABC’D’. Ces travaux ouvrent également de nouvelles voies pour améliorer les performances catalytiques de ces mutants et pour mieux comprendre les déterminants moléculaires et dynamiques qui pourraient jouer un rôle dans la reconnaissance des accepteurs et leur glucosylation sélective, ce qui pourra offrir de nouvelles possibilités pour la synthèse des haptènes de S. flexneri

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

International audienceCombined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

International audienceAmong carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction

Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans

International audienceCarbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerousbiological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by theanomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing indemand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic andchemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD−MS methods for the precise structuralcharacterization of complex glycans

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

International audienceThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio-and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN 123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN 123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the interrelationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens. Carbohydrate-active enzymes catalyze a wide range of chemical reactions. They have emerged as a practical alternative to chemical catalysts, avoiding multiple steps of protection and deprotection often required in chemical synthesis to control the reactivity of the sugar hydroxyl groups and regio-and stereo-selectivity of the reaction. Some of them are rather versatile biocatalysts often displaying naturally a relaxed substrate specificity. This promiscuity can be further exacerbated by enzyme engineering to either broaden or narrow down the range of recognized substrates and/or control the reaction selectivity 1. In particular, mutagenesis targeting the enzym

A convergent chemoenzymatic strategy to deliver a diversity of Shigella flexneri serotype-specific O-antigen segments from a unique lightly protected tetrasaccharide core

International audienceProgress in glycoscience is strongly dependent on the availability of broadly diverse tailored-made, well-defined and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrheal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization post enzymatic -D-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the Glycoside Hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof-of-concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemo-enzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective -D-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest

Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides

International audienceEnzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active α-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens