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Flavonoid Glucodiversification with Engineered Sucrose-Active Enzymes Yannick Malbert

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Flavonoid glucodiversification with engineered sucrose-active enzymes Yannick Malbert To cite this version: Yannick Malbert. Flavonoid glucodiversification with engineered sucrose-active enzymes. Biotechnol- ogy. INSA de Toulouse, 2014. English. NNT : 2014ISAT0038. tel-01219406 HAL Id: tel-01219406 https://tel.archives-ouvertes.fr/tel-01219406 Submitted on 22 Oct 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Last name: MALBERT First name: Yannick Title: Flavonoid glucodiversification with engineered sucrose-active enzymes Speciality: Ecological, Veterinary, Agronomic Sciences and Bioengineering, Field: Enzymatic and microbial engineering. Year: 2014 Number of pages: 257 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 -1 glucose repressor. Using the predicted optimal conditions, 5740 U. L of 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. Keywords: Flavonoid glycosylation, glucansucrase, transglucosylase, Protein engineering, Antioxidant, Glucoconjugates diversification, amylosucrase, α-(1→2) branching sucrose, screening, response surface methodology, enzyme expression optimization. Doctoral school: SEVAB (Ecological, Veterinary, Agronomic Sciences and Bioingineering) Laboratory: Laboratory of Biosystems and Chemical Bioengineering (UMR CNRS 5504, UMR INRA 792), INSA, Toulouse 3 NOM: MALBERT Prénom: Yannick Titre: Glucodiversification des flavonoïdes par ingénierie d’enzymes actives sur saccharose Spécialité: Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieuries, Filière : Ingénieurie microbienne et enzymatique. Année: 2014 Nombre de pages: 257 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 -1 (inducteur) et glucose (répresseur). En appliquant les conditions optimales prédites, 5740 U.L 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. Mots Clés: Glycosylation, flavonoïdes, glucan-saccharase, transglucosylase, ingénierie des protéines, anti-oxidant, diversification de glucoconjugués, amylosaccharase, glucansaccharase de branchement α-(1→2), criblage, méthodologie en response de surface, optimisation de production d’enzyme. Ecole doctorale: SEVAB (Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieuries) Laboratoire: Laboratoire d’Ingénieurie des Systèmes Biologiques et des Procédés (UMR CNRS 5504, UMR INRA 792), INSA, Toulouse 4 PUBLICATIONS Functional and structural characterization of α-(1→2) branching sucrase derived from DSR-E glucansucrase. Brison Y., Pijning T., Malbert Y., Fabre E., Mourey L., Morel S., Potocki-Véronèse G., Monsan P., Tranier S., Remaud-Siméon M., 2012, J. Biol. Chem., 287, 7915–7924. Optimizing the production of an α-(1→ 2) branching sucrase in Escherichia coli using statistical design. Malbert Y., Vuillemin M., Laguerre S., Remaud-Siméon M., and Moulis C., 2014, Appl. Microbiol. Biotechnol., methods and protocol, 1–12. Extending the structural diversity of alpha-flavonoid glycosides with engineered glucansucrases. Malbert Y., Pizzut-Serin S., Massou S., Cambon M., Laguerre S., Monsan P., Lefoulon F., Morel S., André I., and Remaud-Simeon M., 2014, ChemCatChem, Accepted (DOI: 10.1002/cctc.201402144R1) Interactions between sugars and glucan binding domains: structural evidences from the truncated GH70 glucansucrase ∆N123-GBD-CD2. Brison Y., Malbert Y., Mourey L., Remaud-Siméon M. and Tranier S. In preparation. ORAL COMMUNICATIONS Biocatalytic production of flavonic gluco-conjuguates. Malbert
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  • Shilin Yang Doctor of Philosophy

    Shilin Yang Doctor of Philosophy

    PHYTOCHEMICAL STUDIES OF ARTEMISIA ANNUA L. THESIS Presented by SHILIN YANG For the Degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF LONDON DEPARTMENT OF PHARMACOGNOSY THE SCHOOL OF PHARMACY THE UNIVERSITY OF LONDON BRUNSWICK SQUARE, LONDON WC1N 1AX ProQuest Number: U063742 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U063742 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGEMENT I wish to express my sincere gratitude to Professor J.D. Phillipson and Dr. M.J.O’Neill for their supervision throughout the course of studies. I would especially like to thank Dr. M.F.Roberts for her great help. I like to thank Dr. K.C.S.C.Liu and B.C.Homeyer for their great help. My sincere thanks to Mrs.J.B.Hallsworth for her help. I am very grateful to the staff of the MS Spectroscopy Unit and NMR Unit of the School of Pharmacy, and the staff of the NMR Unit, King’s College, University of London, for running the MS and NMR spectra.