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Intitulé du sujet: Interdisciplinary approach for the characterization of secondary metabolites in the filamentous fungus Podospora anserina: to a better understanding of fungal physiology and the isolation of new bioactive metabolites

Sujet

Codirection:

Nombre de mois: 48 mois

Ecole Doctorale: ED 563 - Médicament,Toxicologie, Chimie, Imageries

Unité de recherche et équipe:

CiTCoM UMR CNRS 8038, équipe PNAS

Coordonnées de l’équipe:

4 avenue de l'Observatoire 75006 Paris  

Secteur: Sciences de la vie / Life Sciences

Langue attendue: Anglais

Niveau de langue attendu: B2

Description

Description du sujet:

For human benefits, fungi represent a limitless source of bioactive metabolites that can find applications in pharmaceuticals as exemplified by the antibiotic penicillin. Besides, fungi may also produce dreadful mycotoxins such as gliotoxin, which is a major concern in food industry. From the ecological side, these secondary metabolites play a crucial role for the fungi as they mainly serve as protectors against competitors and help to expand in the environment. However, to date, only a limited number of secondary metabolite production pathways have been elucidated in fungi compared to their potential ability based on genomic mining. Recent availability of genome sequences for a growing number of fungi has led to rapid progress in the identification of genes that are putatively responsible for secondary metabolites production. However, in most cases these gene clusters are silent. One could expect from their activation an access to a large diversity of secondary metabolites. On the other way, metabolomics approaches, using High Resolution Mass Spectrometry is nowadays a convenient tool to efficiently explore natural product secondary metabolites production. It allows description of the metabolome of a species and the comparison of expressed biosynthetic pathways under selected environmental and/or physiological conditions.

Herbivore dungs are a highly competitive habitat where a lot of species are present (as fungi, bacteria, larvae of insects) and feed on partially degraded plant material. It is therefore predictable that coprophilous fungi, as Podospora anserina, are able to notably figure in the chemical war occurring in the inter-species competition, by killing/preventing the growth of competitors using secondary metabolites as weapons. P. anserina possesses a high number of putative secondary metabolites enzymes with 18 polyketide synthase (PKS), 8 nonribosomal peptide synthase (NRPS) and 3 dual PKS/NRPS clusters. The role of sterigmatocystin (ST), an aflatoxin precursor, during the life cycle of P. anserina, in particular during the sexual reproduction and under stress conditions, was investigated by Ling Shen [1] as well as the involvement of the transcriptional regulator NsdD [2]. Recently, Xiaoyue Peng studied how annularin F, a small molecule in the a-pyrone family, was synthesized in P. anserina. (Peng et al in preparation). However, until now, few chemical investigations have been conducted and the contribution of secondary metabolites during the life cycle of P. anserina remains unclear, mainly because most of secondary metabolite clusters are silent or expressed at a very low level in laboratory culture conditions. P. anserina is also an efficient model system because its complete genome sequence is available and annotated, its generation cycle is 2–3 times shorter than other fungi, making it an excellent tool for genetic analyses, and it’s a very efficient organism for transformation to access mutants.

So, in this context, the main objectives of the thesis project are:

(1) activate secondary metabolites gene clusters;

To unlock secondary metabolite clusters that remains usually silent under laboratory culture conditions we propose the incorporation of exogenous chemical factors to enable their expressions, especially by modulating the chromatine state. We will also undertake the overexpression of some specific or known global transcription factors involved in expression of secondary metabolic clusters. This approach will be associated with the construction of deleted strains in genes encoding key enzymes of target clusters and phenotypical characterization of deregulated and mutant strains. Co-cultures of P. anserina with other bacteria or fungi will be also investigated. These approaches will be carried out by the PNAS team (UMR CiTCoM), recently joined by GRR and FCL, who have considerable expertise in fungal genetics experiments, particularly on P. anserina [1–8].

(2) identify and characterize these metabolites;

We will focus on the characterization and comparison of the secondary metabolites production of P. anserina wild type strain and mutants under different growth culture conditions by means of UHPLC-HRMS/MS analyses. This step should allow the identification of induced or deleted secondary metabolites families and pathways. Construction of molecular networks on the basis of the fingerprints obtained from all strain extracts will give insights on the regulation of the metabolome expression, correlating gene clusters presence and observed metabolic pathways. Structure of potentially new compounds will be resolved by use of NMR and MS spectroscopy. All these investigations will be done in that has a long-standing expertise in the isolation and structural elucidation of natural products and also in medicinal chemistry [1,7,9,10]. In addition, the biological profile of isolated compounds should be evaluated at two levels: (1) the influence of these metabolites during the life cycle of P. anserina and also their impact on the growth capacity of other microorganisms; (2) the pharmacological evaluation in the context of potential human applications, this study being externalized to the CIBI Plateform (ICSN, CNRS, Gif-sur-Yvette).

Compétences requises:

The student recruited will have strong experience of working at the microbiology/bio-informatics/chemistry interface and will have a strong interest in fundamental research.
Microbiology: knowledge of filamentous fungi and their metabolism will be a real plus.
Chemistry: sensitivity to the chemistry of natural substances, metabolomics, knowledge of the structures and functions of secondary fungal metabolites.
Bioinformatics: data management, transcriptomics, metabolomics, etc.
Communication: report writing, oral presentations, presentation of results.


Références bibliographiques:

  1. Shen, L.; Porée, F.-H.; Gaslonde, T.; Lalucque, H.; Chapeland-Leclerc, F.; Ruprich-Robert, G. Functional Characterization of the Sterigmatocystin Secondary Metabolite Gene Cluster in the Filamentous Fungus Podospora Anserina: Involvement in Oxidative Stress Response, Sexual Development, Pigmentation and Interspecific Competitions. Environ. Microbiol. 2019, 21, 3011–3026, doi:10.1111/1462-2920.14698.
  2. Shen, L.; Gaslonde, T.; Roullier, C.; Wang, H.; Bodin, J.; Porée, F.-H.; Ruprich-Robert, G.; Chapeland-Leclerc, F. Functional Characterization of the GATA-Type Transcription Factor PaNsdD in the Filamentous Fungus Podospora Anserina and Its Interplay with the Sterigmatocystin Pathway. Appl Environ Microbiol 2022, aem0237821, doi:10.1128/aem.02378-21.
  3. Xie, N.; Chapeland-Leclerc, F.; Silar, P.; Ruprich-Robert, G. Systematic Gene Deletions Evidences That Laccases Are Involved in Several Stages of Wood Degradation in the Filamentous Fungus Podospora Anserina. Environmental Microbiology 2014, 16, 141–161, doi:10.1111/1462-2920.12253.
  4. Xie, N.; Ruprich-Robert, G.; Chapeland-Leclerc, F.; Coppin, E.; Lalucque, H.; Brun, S.; Debuchy, R.; Silar, P. Inositol-Phosphate Signaling as Mediator for Growth and Sexual Reproduction in Podospora Anserina. Dev. Biol. 2017, 429, 285–305, doi:10.1016/j.ydbio.2017.06.017.
  5. Xie, N.; Ruprich-Robert, G.; Silar, P.; Herbert, E.; Ferrari, R.; Chapeland-Leclerc, F. Characterization of Three Multicopper Oxidases in the Filamentous Fungus Podospora Anserina: A New Role of an ABR1-like Protein in Fungal Development? Fungal Genet. Biol. 2018, 116, 1–13, doi:10.1016/j.fgb.2018.04.007.
  6. Shen, L.; Chapeland-Leclerc, F.; Ruprich-Robert, G.; Chen, Q.; Chen, S.; Adnan, M.; Wang, J.; Liu, G.; Xie, N. Involvement of VIVID in White Light-Responsive Pigmentation, Sexual Development and Sterigmatocystin Biosynthesis in the Filamentous Fungus Podospora Anserina. Environmental Microbiology 2022, 24, 2907–2923, doi:10.1111/1462-2920.15978.
  7. Shen, L.; Roullier, C.; Porée, F.-H.; Gaslonde, T.; Riffault-Valois, L.; Grovel, O.; Ruprich-Robert, G.; Chapeland-Leclerc, F. Complementary Strategies to Unlock Biosynthesis Gene Clusters Encoding Secondary Metabolites in the Filamentous Fungus Podospora Anserina. Journal of Fungi 2023, 9, 9, doi:10.3390/jof9010009.
  8. Essadik, I.; Boucher, C.; Bobée, C.; Cabet, É.; Gautier, V.; Lalucque, H.; Silar, P.; Chapeland-Leclerc, F.; Ruprich-Robert, G. Mutations in Podospora Anserina MCM1 and VelC Trigger Spontaneous Development of Barren Fruiting Bodies. J Fungi (Basel) 2024, 10, 79, doi:10.3390/jof10010079.
  9. Gaslonde, T.; Gadea, A.; Khazem, M. Current Knowledge on Chemistry of Proteaceae Family, and Biological Activities of Their Bis-5-Alkylresorcinol Derivatives. Phytochemistry Reviews 2022, doi:10.1007/s11101-022-09821-4(0123456789().,-volV().
  10. Guedes da Silva Almeida, J.R.; Dutra, L.M.; dos Anjos Santos, V.L.; Rolim, L.A.; Lopes, N.P.; Gaslonde, T.; de Oliveira-Júnior, R.G.; Grougnet, R. Flavonoids from Passiflora Cincinnata (Passifloraceae): HPLC-DAD-MSn Characterization, Validation of an HPLC-DAD Method for Quantification and CPC Isolation. Phytochemistry Letters 2024, 62, 84–91, doi:10.1016/j.phytol.2024.06.006.