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Дисертації з теми "Champignons endophytes – Cultures cellulaires":
Vassaux, Antoine. "Mécanisme de biosynthèse et production de l’astine, un pentapeptide cyclique non-ribosomique de Cyanodermella asteris." Electronic Thesis or Diss., Université de Lille (2018-2021), 2019. http://www.theses.fr/2019LILUR027.
Astin C is a cyclic peptide assembled through a nonribosomal mechanism by a NonRibosomal Peptide Synthetase (NRPS). This nonribosomal peptide displays promising therapeutic properties including anti-tumor and anti-inflammatory activities. For decades, this compound was only extracted from the roots of Aster tataricus, a well-known plant in traditionnal japanese and chinese medicine. Recently, Cyanodermella asteris, a fungal endophyte of this plant, was demonstrated to be able to synthesize astin C. This discovery offers new opportunities for the production of this compound of interest. Nonetheless the very low growth rate of this endophytic fungus is an obstacle limiting the astin C production. In this study, two distinct approaches were conjointly considered to upscale the production rate of this compound. The first strategy was related to an optimization of the homologous production from C. asteris. For this purpose, an innovative cultivation system has been developed enabling to grow the fungus exclusively on a stainless steel support. This cultivation condition turned out to be favorable both for the fungal biomass development and for the astin C production. In order to further optimize this process, the effects of several culture parameters (i.e. support pre-treatment procedure, inoculum type, pH, medium composition) on the astin C production rates was investigated. Under optimized conditions, astin C yields were further enhanced, constituting a first step towards the development of an astin C production process at industrial scale. Meanwhile, a study was conducted in order to develop a heterologous expression system for astin C production in yeast. The identification, through bioinformatics analyses, of the genes involved in the astin biosynthesis was a precondition for the development of this approach. Once these genes have been identified, a literature review has enabled to compile the molecular tools applicable for the cloning of NRPS long lenght sequence, and to select a proper host to heterologously express them. The whole sequence or a truncated one have been transfered respectively to Saccharomyces cerevisiae or Yarrowia lipolytica. In boh considered yeasts, a heterologous expression of the foreign sequences was confirmed. In S. cerevisiae, the synthesis of the astin NRPS could not be demonstrated. On the other hand, in Y. lipolytica, for the first time, the production of a NRPS-type structure was detected. Although no nonribosomal peptide was detected, this study enabled to overcome several of the hurdles limiting the development of a astin heterologous production way in yeast
Mephane, Eléonore. "Conception de cocktails issus de co-cultures de bactéries et champignons pour de nouveaux bio-fongicides." Electronic Thesis or Diss., Université de Lille (2022-....), 2022. https://pepite-depot.univ-lille.fr/ToutIDP/EDSMRE/2022/2022ULILR074.pdf.
The genus Fusarium causes plant pathologies affecting a wide variety of targets with consequences on yields and consumer health. Among them, F. graminearum and F. oxysporum have the most important economic impacts and sustainable control methods against these pathogens are currently limited. Biocontrol is an alternative to synthetic pesticides. However, it is difficult to fully exploit the potential that exists in nature. One way to discover new molecules of interest is co-culture. Involving two or more populations of cells, it recreates interactions that do not exist in monocultures. The aim of this thesis project was to bring together bacteria and fungi with known activities, and thus to discover associations producing cocktails of antifungal molecules to fight against phytopathogens.The project started with the rational selection of microorganisms with antifungal activity reported in the literature: five bacteria (Bacillus subtilis, Pseudomonas syringae, Dietzia sp., Streptomyces coelicolor, Streptomyces sp.) and five fungi (Pseudozyma aphidis, Trichoderma harzianum, Aspergillus oryzae, Cladosporium cladosporioides, and F. oxysporum) were chosen. Subsequently, culture conditions (medium, temperature) adequate to perform co-cultures and allow the growth of both partners involved were defined and three media were chosen: two rich media (LB, NB) and one minimal medium (GMM).After this selection, tests in a microbioreactor (BioLector) were carried out: these involved the ten selected microorganisms in monocultures and twenty-five co-cultures in the three media conditions. A screening of the antifungal activity of the generated culture supernatants was performed against an environmental strain of F. oxysporum and S. cerevisiae. Thirteen out of twenty-five co-cultures showed activity against at least one of the two targets. After these tests, the selection of co-cultures of interest was reduced from twenty-five to ten. These co-cultures were grown in 50 mL volumes in LB and NB media that showed the best activity under the chosen conditions, and their supernatants tested for antifungal activity. These tests allowed to refine the choice and to focus on six couples: P. syringae + A. oryzae, Streptomyces sp. + A. oryzae, P. syringae + F. oxysporum, P. syringae + P. aphidis, Dietzia sp. + T. harzianum, Streptomyces sp. + C. cladosporioides. These six couples were subjected to a series of cultures and their supernatants tested on agar plates and in liquid media (against F. oxysporum). Three co-cultures showed a more pronounced activity, especially against F. oxysporum and stood out from the monocultures: Streptomyces sp. + A. oryzae, Streptomyces sp. + C. cladosporioides and P. syringae + A. oryzae. The Streptomyces sp. + C. cladosporioides co-culture showed synergistic activity in inhibiting or slowing the growth of F. oxysporum compared to monocultures alone, while P. syringae + A. oryzae and Streptomyces sp. + A. oryzae showed additive activity against F. oxysporum.For the three selected couples, the molecules produced and secreted were studied by proteomics and metabolomics. Whatever the co-culture considered, it induces the activation of genes that remained silent in monoculture. Thus, we can observe the expression of a very high proportion of proteins or secondary metabolites (38 to 50%) exclusively present in the supernatants of co-cultures. Moreover, among the molecules secreted de novo in the co-cultures, some known for their antimicrobial or even antifungal activities could be identified for the three couples that were studied
Malbreil, Mathilde. "La biologie du champignon mycorhizien à arbuscules Rhizophagus irregularis DAOM 197198 à la lumière de la génomique et de la transcriptomique." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2670/.
The biology of the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM197198 enlighten by genomics and transcriptomics. Glomeromycota are mutualistic fungi associated with plant roots. The vast majority of plant species are able to form a symbiosis with these organisms. This association improves plant nutrition via a better water and mineral recruitment from the soil. In return, the fungus receives carbon compounds. Symbiosis establishment is achieved by a step by step development, led by signal exchanges. My work was focused on describing genetic programs supporting the fungal development of Rhizophagus irregularis DAOM197198, during symbiosis establishment. The fungus was grown in several conditions representing key point of its development, and RNA were sequenced by illumina. RNAseq data obtained were then used to define the gene models on the genome assembly. R. Irregularis has a haploid and homocaryotic genome (Tisserant*, Malbreil* et al. , 2013). Strigolactones (plant signal molecules) affect the expression of around 300 genes during the pre-symbiotic development, in a sequential manner. By studying the R. Irregularis transcriptomes in association with 3 phylogenetically distant plants, we report that a set of 262 genes are highly induced whatever the host is. These results allowed refining the number of candidate genes possibly playing an important role in the symbiotic development. Finally, by coupling metabolomic and transcriptomic approaches, two molecules (propionyl- and butyryl-carnitine) could be identified and might play a role in late steps of symbiosis establishment
Triastuti, Asih. "Exploration de la diversité chimique dans les endophytes fongiques : influence de l'addition des modificateurs épigénétiques et des co-cultures fongiques sur le métabolome de Botryosphaeria mamane." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30228.
This study focused on the strain of a poorly studied fungal endophyte Botryosphaeria mamane E224, isolated from Bixa orellana leaves. Our previous screening involving 409 fungal strains isolated from medicinal plants from South America revealed that among all these strains, B. mamane was shown to be the most bioactive on in vitro model against Leishmania infantum. The objectives of this work consisted in the introduction of new metabolite production by B. mamane by optimizing the fungal culture conditions, and by using co-cultivation methods and addition of epigenetic modifiers. This work was followed by an analysis of the different metabolomes via a metabolomics approach using UHPLC-HRMS and integration of informatics and statistical tools for metabolomics. Two major compound classes were detected in B. mamane. First, the cyclopeptide family including the thiodiketopiperazines (TDKPs) alkaloids with three new compounds proposed as botryosulfuranols A-C; and the isocoumarin family, with the mellein derivatives, trans-4-hydroxymellein, 4-hydroxymellein, and 5-hydroxymellein. Regarding the exploration of B. mamane metabolome cultured in the presence of epigenetic modifier, the effects of two different histone deacetylase inhibitors (HDACis), suberoylanilidehydroxamic acid (SAHA) and valproate sodium added in two different stages of fungal growth, were investigated. As expected, HDACis addition in the culture of B. mamane led to significant changes in the secondary metabolite production. Addition of modifier not only induced metabolites production but also reduced and may inactivate metabolite production in fungi, depending on the nature of the epigenetic modifier added. This study illustrates the importance in the choice of HDACis to fungal culture in order to induce specific metabolite productions. In the study of B. mamane and C. albicans co-cultivation in different culture conditions, we showed the influence of the conditions (static versus agitation) on the metabolome of the fungi. However, the co-culture with yeast did not induce any modification in the fungal metabolome. The investigation of fungal interactions between B. mamane, Fusarium solani, and Colletotrichum linicola in 6-multi well plates in time-series based analysis has been carried out. [...]
Chausse, Rémi. "Recherche d'enzymes ligninolytiques dans la biodiversité, mise en évidence d'enzymes de type oxydoréductase provenant de l'ascomycète Scedosporium apiospermum." Electronic Thesis or Diss., Université de Lille (2022-....), 2022. http://www.theses.fr/2022ULILR077.
AbstractThe goal of this thesis was to search for new fungal ligninolytic enzymes from the environment. Samples of woody biomass were collected in three different ecosystems: a forest composed of deciduous trees (beech, birch) and gymnosperm (Cupressus macrocarpa), a cep of chardonnay infected with grapevine trunk disease and finally a decaying tree stump in a parking lot of an urban area. 71 strains were isolated from these environments. To select the fungal strain able to produce enzymes of interest a screening for laccase, Lip and Mnp was carried out. In order to maximize the completeness of this screening different culture conditions were used. To trigger the production of ligninolytic enzymes by the fungi Three culture media supplemented or not with inductors were used for the screening. The first medium tested was a rich medium without induction. The two other media contained respectively wheat straw and guaiacol as inductors. Besides, sampling was performed after 7 and 14 days as the kinetics of enzyme production could vary for a given fungal strain and culture conditions. 71 strains were tested for ligninolytic activities and 15 of them presented at least one enzymatic activity. Two of them showed their ligninolytique activities only on the media with inductors. Among the 15 fungi with enzymatic activities of interest, two basidiomycetes and 13 ascomycetes belonging to 6 different genera were identified. 12 ascomycetes were already described in the literature with laccase, Lip or Mnp activity. To our knowledge, the two basidiomycetes Peniophora versicolor and Piptoporus betulinus were not described for producing ligninolytic activities. Besides Scedosporium apiospermum showed laccase activity during the screening. Experiments of lignin degradation were carried out with the culture supernatant of this fungus. The results showed that the enzymes contained in S. apiospermum were able to modify the lignin polymer. This fungus was thus selected to further characterize its laccase like enzymes detected by the screening. Thanks to an in-silico screening in the genome of S. apiospermum 9 putative laccase genes were detected. Five putative laccase genes were then amplified with success from a cDNA library obtained from the mRNA of the fungus. Those five genes were cloned in the yeast Yarrowia lipolytica. In the conditions of the study, two genes called Lac2 and ITMO2 allowed the production of two active enzymes. In their primary structure, the two enzymes present the characteristic sites of copper coordination of multicopper oxidases. Lac2 and ITMO2 present an optimum pH of 3 and 5 respectively. Those two enzymes show an oxidative activity on various phenolic substrates such as 2.6 dimetoxyphenol, pyrogallol and dopamine but also on non-phenolic substrates including 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and o-Dianisidine
Vassaux, Antoine. "Mécanisme de biosynthèse et production de l’astine, un pentapeptide cyclique non-ribosomique de Cyanodermella asteris." Thesis, Lille 1, 2019. http://www.theses.fr/2019LIL1R027/document.
Astin C is a cyclic peptide assembled through a nonribosomal mechanism by a NonRibosomal Peptide Synthetase (NRPS). This nonribosomal peptide displays promising therapeutic properties including anti-tumor and anti-inflammatory activities. For decades, this compound was only extracted from the roots of Aster tataricus, a well-known plant in traditionnal japanese and chinese medicine. Recently, Cyanodermella asteris, a fungal endophyte of this plant, was demonstrated to be able to synthesize astin C. This discovery offers new opportunities for the production of this compound of interest. Nonetheless the very low growth rate of this endophytic fungus is an obstacle limiting the astin C production. In this study, two distinct approaches were conjointly considered to upscale the production rate of this compound. The first strategy was related to an optimization of the homologous production from C. asteris. For this purpose, an innovative cultivation system has been developed enabling to grow the fungus exclusively on a stainless steel support. This cultivation condition turned out to be favorable both for the fungal biomass development and for the astin C production. In order to further optimize this process, the effects of several culture parameters (i.e. support pre-treatment procedure, inoculum type, pH, medium composition) on the astin C production rates was investigated. Under optimized conditions, astin C yields were further enhanced, constituting a first step towards the development of an astin C production process at industrial scale. Meanwhile, a study was conducted in order to develop a heterologous expression system for astin C production in yeast. The identification, through bioinformatics analyses, of the genes involved in the astin biosynthesis was a precondition for the development of this approach. Once these genes have been identified, a literature review has enabled to compile the molecular tools applicable for the cloning of NRPS long lenght sequence, and to select a proper host to heterologously express them. The whole sequence or a truncated one have been transfered respectively to Saccharomyces cerevisiae or Yarrowia lipolytica. In boh considered yeasts, a heterologous expression of the foreign sequences was confirmed. In S. cerevisiae, the synthesis of the astin NRPS could not be demonstrated. On the other hand, in Y. lipolytica, for the first time, the production of a NRPS-type structure was detected. Although no nonribosomal peptide was detected, this study enabled to overcome several of the hurdles limiting the development of a astin heterologous production way in yeast
Yammine, Marie. "Caractérisation glycoprotéomique des mannoprotéines de la paroi cellulaire de la levure Saccharomyces cerevisiae : comparaison des parois natives et après fractionnement industriel." Electronic Thesis or Diss., Université de Lille (2018-2021), 2021. http://www.theses.fr/2021LILUR067.
Yeast cell wall is the outermost organelle of the yeast cell. It composed of an inner layer of polysaccharides, consisting of β-glucans mostly cross-linked to a minor amount of chitin, and to which are linked mannoproteins by covalent or non-covalent bonds. Mannoproteins form the outer layer of the yeast cell wall and are considered as its second most abundant component. Yeast cell wall mannoproteins are proteins that are highly mannosylated by short simple O-glycans and by large complex N-glycans. They have particular functional properties and exceptional nutritional value related to their molecular structure, but have been little investigated. This work aims to study yeast cell wall mannoproteins at the molecular level by different techniques based on high resolution mass spectrometry.The mannoproteins were extracted by physical, chemical methods eventually coupled to enzymatic methods from the reference strain S288C grown in different modes in bioreactors in the presence of a culture medium, or from industrial yeast samples, or from already fractionated industrial yeast products. These mannoproteins were then O- and N-deglycosylated chemically or enzymatically. The resulting peptides were analyzed by nanoESI-LC-MS/MS. Bioinformatics analysis of these data allowed the identification and quantification of mannoproteins using Saccharomyces cerevisiae S288C reference strain database. The deglycosylation of the peptides was also verified. Gene ontology analysis was then performed to determine the subcellular location of the identified proteins. The O- and N-glycans were chemically derivatized by a reductive amination reaction and then analyzed by µESI-LC-MS and capillary electrophoresis respectively.This work allowed us to compare different methods of extraction of mannoproteins from the yeast cell wall. These different methods result in qualitative or quantitative enrichment of different types of mannoproteins and in the presence of other proteins mainly annotated as being related to organelle membranes (especially mitochondrial and nuclear). Further purification of wall isolates was applied to reduce the number of these non-cell wall proteins. The development of an enzymatic N-deglycosylation protocol using a one-pot method without sample transfer allowed an increase in the coverage of identified mannoproteins. Using other enzymes, the same protocol allows a gentle O-deglycosylation but degrades O-glycans into monosaccharides. Enzymatic N-deglycosylation combined to chemical O-deglycosylation allows the simultaneous isolation of O- and N-glycans from mannoproteins, allowing their subsequent analysis by mass spectrometry and capillary electrophoresis after chemical derivatization with appropriate labels by reductive amination reaction. The protein profiles differ qualitatively and quantitatively according to the growth phase and culture mode. We identified some of their protein markers, which are markers of glucose deprivation expressed in the stationary growth phase of batch and fed batch culture.This glycoproteomic approach was also applied to the glycoproteomic and peptidomic characterization of products generated by different industrial processing methods, intended for commercial use, allowing to decipher their complex nature in terms of composition and structure