La voie de synthèse non-ribosomique représente une source importante de molécules d’intérêt. Mettant en jeu de larges complexes multi-enzymatiques, les Synthétases de Peptides Non Ribosomiques (NRPS), ce mode de production aboutit à la formation de peptides originaux d’une grande diversité structurale et fonctionnelle. Les problématiques de recherche de nouvelles molécules d’origines naturelles pour une utilisation dans différents domaines industriels font des molécules d’origine non-ribosomique de parfaites candidates pour la découverte des médicaments, surfactants ou antibiotiques des prochaines années. Les travaux décrits ont pour but l’étude du mécanisme de production de deux familles de molécules issues de la synthèse non-ribosomique, la kurstakine, un lipopeptide produit par Bacillus thuringiensis, et les PPCC (PentaPeptides Cycliques Chlorés), regroupant plusieurs molécules d’origine eucaryote. L’étude de la production de kurstakine par une série de mutants de B. thuringiensis a permis d’identifier plusieurs facteurs limitant la production de cette molécule, et d’ouvrir la voie à la mise en place de procédé de production en milieu liquide de cette molécules. L’étude des PPCC a permis d’identifier plusieurs gènes NRPS potentiellement impliqués dans la production de ces familles de molécules dans le génome de deux moisissures. / The Non Ribosomal Synthesis pathway is a very important source of molecules of interest. Based on large multi-enzymatic complexes, the Non Risosomal Peptide Synthetases (NRPS), this production mode leads to the formation of original peptides, with a large structural and activities diversity. The need of new molecules from natural sources, turns the non-ribosomal molecules into a promising source of drugs, surfactant or antibiotics for the future. This work aim to the understanding of the production mechanism of two families of non-ribosomal molecules, the kurstakin, a lipopeptide produced by Bacillus thuringiensis, and the CCPPs (Chlorinated Cyclic Penta Peptides), regrouping several eukaryotic molecules. The study of the kurstakin production using a series of B. thuringiensis mutants led to the identification of several limitating factors for the production of this molecule, and opened the possibility to the setting up of production process in liquid medium of this molecule. The study of CCPPs led to the identification of several genes, potentially implied on the production of these molecules in the genome of two fungal strains.

L’astine C est un peptide cyclique assemblé au cours d’un mécanisme nonribosomique par une synthétase dite NRPS (NonRibosomal Peptide Synthetase). Ce peptide nonribosomique possède des propriétés thérapeutiques intéressantes avec notamment des activités anti-tumorale et anti-inflammatoire. Jusqu’ici ce composé était exclusivement extrait à partir des racines d’Aster tataricus, une plante utilisée traditionnellement en médecine japonaise et chinoise. Récemment, une production d’astine C a été mise en évidence chez Cyanodermella asteris, un champignon endophyte de cette plante. Cette découverte ouvre de nouvelles perspectives en matière de production d’astine C, qui reste néanmoins très limitée en raison du faible taux de croissance du champignon endophyte. Au cours de cette étude, deux approches ont été développées parallèlement afin d’augmenter les taux de production de l’astine C. La première stratégie consistait à augmenter les rendements en optimisant la production homologue à partir de C. asteris. Dans cette optique, un système de culture a été établi afin de cultiver le champignon exclusivement sur un support en acier inoxydable. Ce mode de culture a favorisé à la fois le développement de la biomasse fongique et la production du composé d’intérêt. En vue d’optimiser ce procédé, l’impact de plusieurs paramètres de culture (modalité de préparation du support, type d’inoculum, pH de culture, et composition du milieu de culture) sur la production d’astine C a été évalué. Les paramètres de culture optimisés ont permis d’améliorer de nouveau les rendements en astine C, ce qui constitue une première étape dans le développement d’un procédé de production à l’échelle industrielle. En parallèle, des travaux ont été menés afin de développer un système de production hétérologue d’astine C chez la levure. Cette approche n’a pu être considérée qu’après avoir identifié, par le biais d’analyses bioinformatiques, les gènes impliqués dans la voie de biosynthèse de l’astine. Une fois ces gènes identifiés, une revue de la littérature a permis, entre autres, de dresser un bilan des outils moléculaires disponibles pour le clonage des larges séquences nucléiques codant pour les NRPSs, et de sélectionner des hôtes hétérologues appropriés. Des séquences complète ou partielle du gène codant pour l’astine synthétase ont été clonées respectivement chez Saccharomyces cerevisiae et Yarrowia lipolytica. Pour les deux levures considérées, une expression hétérologue a été constatée. Chez S. cerevisiae, la synthèse de la NRPS d’astine n’a pas pu être démontrée. En revanche, pour la première fois, la production d’une structure de type NRPS chez Y. lipolytica a pu être mise en évidence. Bien qu’aucun peptide nonribosomique n’ait été détecté, cette étude a permis de lever une partie des verrous limitant le développement d’un mode de production hétérologue d’astine chez la levure. / 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.

Aster tataricus (Asteraceae) is a plant native to Northern Asia and known for its use in the Traditional Chinese and Japanese Medicine. Beside many other secondary metabolites, it contains pentapeptides called astins from which some show an antitumor activity against different human cell lines. Astins are chlorinated, cyclic pentapeptides consisting of proteinogenic and non-proteinogenic amino acids. The astin structure indicates the involvement of non ribosomal peptide synthetases as well as flavin-dependent halogenases. Both enzymes are currently only known from bacteria and fungi. A new endophytic fungus Pelliciarosea asterica was isolated from A. tataricus which produces some of the astins found in the different plant organs. The nearest neighbors of P. asterica are ostropalean fungi from the Stictidaceae lineage (Stictidaceae, Ostropales, Lecanoromycetes, Pezizomycetes, Ascomycota). P. asterica is located in all plant organs of A. tataricus but the highest accumulation of the fungus is found in rhizomes and above-ground organs like leaves or inflorescences. In contrast, the highest astin concentration was found in the roots where nearly no fungus was detectable. P. asterica produces only one of the dichlorinated astins (astin C) in liquid culture, but in A. tataricus all three forms of the dichlorinated astins (A/B and C) were found. This indicates that either the plant is “using” the fungal astin C and metabolize it into one of the other astins or that the fungus, once living inside the plant, is itself producing the other astins. It was also searched for a candidate gene of a halogenase which is essential for the dichlorination of the astins with an antitumor activity. No halogenase could be found by PCR or Southern as well as colony blot, neither in A. tataricus nor in P. asterica. Even the genome sequencing of P. asterica revealed no candidate gene for a halogenase. Endophytes support the plant by suppressing pathogens (antibiosis) or by providing additional nutrients like phosphates or iron to the plant. P. asterica can solubilize different phosphate sources on agar plates. Different fungi are inhibited in growth by P. asterica on agar plates. The endophyte P. asterica from A. tataricus supports its host in different ways and produces secondary metabolites. These secondary metabolites seem to be fungal metabolites either used or degraded by the plant. P. asterica is therefore a good alternative for a possible large-scale production of such antitumor acting astins.

Aster tataricus (Asteraceae) is a plant native to Northern Asia and known for its use in the Traditional Chinese and Japanese Medicine. Beside many other secondary metabolites, it contains pentapeptides called astins from which some show an antitumor activity against different human cell lines. Astins are chlorinated, cyclic pentapeptides consisting of proteinogenic and non-proteinogenic amino acids. The astin structure indicates the involvement of non ribosomal peptide synthetases as well as flavin-dependent halogenases. Both enzymes are currently only known from bacteria and fungi. A new endophytic fungus Pelliciarosea asterica was isolated from A. tataricus which produces some of the astins found in the different plant organs. The nearest neighbors of P. asterica are ostropalean fungi from the Stictidaceae lineage (Stictidaceae, Ostropales, Lecanoromycetes, Pezizomycetes, Ascomycota). P. asterica is located in all plant organs of A. tataricus but the highest accumulation of the fungus is found in rhizomes and above-ground organs like leaves or inflorescences. In contrast, the highest astin concentration was found in the roots where nearly no fungus was detectable. P. asterica produces only one of the dichlorinated astins (astin C) in liquid culture, but in A. tataricus all three forms of the dichlorinated astins (A/B and C) were found. This indicates that either the plant is “using” the fungal astin C and metabolize it into one of the other astins or that the fungus, once living inside the plant, is itself producing the other astins. It was also searched for a candidate gene of a halogenase which is essential for the dichlorination of the astins with an antitumor activity. No halogenase could be found by PCR or Southern as well as colony blot, neither in A. tataricus nor in P. asterica. Even the genome sequencing of P. asterica revealed no candidate gene for a halogenase. Endophytes support the plant by suppressing pathogens (antibiosis) or by providing additional nutrients like phosphates or iron to the plant. P. asterica can solubilize different phosphate sources on agar plates. Different fungi are inhibited in growth by P. asterica on agar plates. The endophyte P. asterica from A. tataricus supports its host in different ways and produces secondary metabolites. These secondary metabolites seem to be fungal metabolites either used or degraded by the plant. P. asterica is therefore a good alternative for a possible large-scale production of such antitumor acting astins.