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Analyses Génomiques Et Recherche De Production De Molécules

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Analyses Génomiques Et Recherche De Production De Molécules Analyses génomiques et recherche de production de molécules naturelles antimicrobiennes d’une souche de Streptomyces fulvissimus chez l’hôte natif et par transfert hétérologue chez un hôte alternatif Mémoire Xavier Murphy Després Maîtrise en biochimie - avec mémoire Maître ès sciences (M. Sc.) Québec, Canada © Xavier Murphy Després, 2019 Analyses génomiques et recherche de production de molécules naturelles antimicrobiennes d’une souche de Streptomyces fulvissimus chez l’hôte natif et par transfert hétérologue chez un hôte alternatif Mémoire de Maîtrise en biochimie Xavier Murphy Després Sous la direction de : Manon Couture directrice de recherche Rong Shi, codirecteur de recherche Résumé Développé dans le contexte mondial de lutte aux souches microbiennes résistantes aux antibiotiques, ce projet de maîtrise a pour but d’isoler et de caractériser un opéron de synthèse d’une molécule naturelle potentiellement bioactive, un tétramate polycyclique macrolactame (PTM), provenant d’une souche de streptomycète peu caractérisée mais dont le génome a été récemment séquencé1. L’objectif principal est de transférer l’opéron dans une souche spécialisée de Escherichia coli pour son expression et sa caractérisation. Nous avons observé que la souche d’intérêt, Streptomyces fulvissimus ATCC27431 / DSM 40593, produisait effectivement une molécule capable d’inhiber la croissance d’une levure. Nous n’avons cependant pas été en mesure de réaliser l’isolation et le transfert hétérologue de l’opéron, malgré l’utilisation de trois approches différentes. La première approche consistait à obtenir directement les gènes de l’opéron via une amplification PCR à partir d’ADN génomique de S. fulvissimus. La seconde approche comportait l’assemblage de gènes synthétisés chimiquement pour reconstituer l’opéron sur deux plasmides. La dernière approche impliquait la création puis le criblage d’une librairie d’ADN génomique de S. fulvissimus basée sur le fosmide pCC1FOS dans l’hôte E. coli. Les résultats inattendus issus du séquençage de quelques fosmides ont justifié le séquençage complet de l’ADN génomique de la souche ATCC 27431 / DSM 40593. Les contigs obtenus montrent que notre souche diffère de la souche de S. fulvissimus dont le génome a été publié, ce qui suggère que le génome publié a été incorrectement associé à cette souche. Nos résultats montrent également que l’opéron de biosynthèse du PTM n’est pas présent, en tout ou en partie, dans ces contigs et que ces derniers ne s’alignent pas parfaitement avec l’ADN d’aucune souche connue de streptomycète, ce qui implique que nous avons séquencé, pour la première fois, l’ADN de cette souche de S. fulvissimus. L’analyse bio-informatique des contigs nous a permis de mettre en évidence chez cette bactérie des voies de biosynthèse de molécules naturelles potentiellement bioactives qui pourraient être d’intérêt pour des études futures. iii Abstract In the context of the worldwide fight against drug-resistant microbes, this project aims to isolate and characterize a putative gene cluster for the synthesis of a bioactive molecule, a polycyclic tetramate macrolactam (PTM), from a poorly characterized but recently sequenced strain of Streptomyces1. The main goal is to transfer the gene cluster in a specialised strain of Escherichia coli for its expression and characterization. We observed that the strain of interest, identified as Streptomyces fulvissimus DSM 40593 / ATCC 27431, indeed synthesized a molecule capable of inhibiting the growth of yeast. However, we have not been able to isolate or transfer the gene cluster even though we employed three different strategies. The first strategy involved PCR amplification of the gene cluster directly from S. fulvissimus’ genomic DNA. The second strategy involved the chemical synthesis and enzymatic assembly of the gene cluster on two plasmids. The final strategy involved the construction and screening of a S. fulvissimus genomic DNA library with the pCC1FOS fosmid in the E. coli host. DNA sequencing of a few fosmids generated unexpected results and justified the sequencing of the complete genome of Streptomyces fulvissimus ATCC 27431 / DSM 40593. The assembled DNA contigs differed from the sequence of the Streptomyces strain whose genome was previously published and suggest that the latter was probably mislabeled. Our contigs show that the PTM biosynthetic gene cluster is absent from our strain’s genome, and they do not align perfectly with the sequence of any published genome, which suggests that we sequenced the genome of the S. fulvimissus strain ATCC 27431 / DSM 40593 for the first time. Bioinformatics analysis allowed the detection of genes potentially involved in the biosynthesis of bioactive molecules that could be of interest in future studies. iv Table des matières Résumé ............................................................................................................................. iii Abstract ............................................................................................................................. iv Table des matières ............................................................................................................ v Liste des figures ................................................................................................................. ix Liste des tableaux .............................................................................................................. x Liste des abréviations ........................................................................................................ xi Remerciements ................................................................................................................ xiii Introduction ........................................................................................................................ 1 Problématique ................................................................................................................ 1 Les antibiotiques et l’apparition de la résistance aux antibiotiques .............................. 1 La résistance aux antimicrobiens ................................................................................ 2 Les mécanismes de résistance chez les microbes ...................................................... 3 L’approche Top-down : Les approches traditionnelles de découverte des antibiotiques ................................................................................................................................... 5 Des nouvelles approches en découverte d’antibiotiques et en thérapie antimicrobienne .......................................................................................................... 6 Le séquençage de nouvelle génération et le potentiel de découvertes biologiques ..... 8 Les avancées en métagénomique .............................................................................. 9 Les grandes approches en métagénomique ............................................................. 10 Le criblage fonctionnel d’échantillons métagénomiques ............................................ 11 Cadre théorique ........................................................................................................... 12 Le transfert hétérologue d’ADN ................................................................................. 12 La construction de plasmides via une amplification PCR de gènes d’intérêt ............. 12 La synthèse chimique de gènes ................................................................................ 13 Principes généraux ................................................................................................... 13 L’optimisation des codons des gènes ....................................................................... 13 Les librairies génomiques basées sur les fosmides .................................................. 14 Les Streptomycètes .................................................................................................. 15 Streptomyces fulvissimus et son génome ................................................................. 15 Les Polycyclic Tetramate Macrolactams (PTM) ........................................................ 17 Les polycétides synthases (PKS) .............................................................................. 18 Les synthétases de peptides non-ribosomaux (NRPS) ............................................. 20 Les PKS/NRPS hybrides et les PTM ......................................................................... 22 v Le transfert hétérologue des PKS/NRPS .................................................................. 24 Les gènes codant le PTM de Streptomyces fulvissimus DSM 40593 ........................ 25 Les enzymes de décoration de l’opéron PTM ........................................................... 27 Hypothèse et objectifs : ................................................................................................ 28 Hypothèse ................................................................................................................ 28 Objectifs de recherche .............................................................................................. 28 Notes sur les stratégies de réalisation du projet ........................................................ 28 Clonage par amplification PCR ................................................................................. 28 Synthèse chimique des gènes .................................................................................
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