Integrated physiology and functional genomics of yeast and filamentous fungi (PHYGE)

Team responsables: Jean Marie FRANCOIS & Jean Luc PARROU

 

Jean Marie FRANCOIS got his PhD from University of Louvain in 1988 (Belgium). He was appointed professor of Industrial Microbiology and BioNanotechnology at the National Institute of Applied Sciences, University federal of Toulouse, in 1993 and reached the Exceptional class in 2009. He is member of the Microbial physiology and Biotechnology & Bioengineering section of the European Federation of Biotechnology. He is editor in chief in BMC Biotechnology for Biofuels since 2017, editor associated of Frontiers in Synthetic Biology, member of the editorial board of FEMS Yeast Research and of Microbial Cell. He has been appointed since 2016 as expert member of European Science Foundation. He holds more than 180 publications in international refereed journals and 17 patents. In 2010, he is the co-founder of the Dendris company which innovates in molecular in vitro diagnostic by exploiting recent development in Nanotechnology in which is was involved through his implication in the creation of the “Institut Avancées des Sciences du Vivant” (ITAV) in 2009. 

Jean Luc PARROU got his PhD from the INSA Toulouse, in 1996. After a post-doc in D. Shore’s lab in the Department of Molecular Biology from Geneva University, he got in 2000 a position as CNRS researcher, in Pr JM François’ lab, now leading the research topic number 2 of the team. He is responsible of the PCR and qPCR teaching modules given in the frame of continuing education (“Toulouse Tech Formation Professionelle”). He held in 2012 the organization of the 10th francophone yeast meeting (LMO10), and created the YFFnet website, dedicated to communication in the ‘yeasters’ community. Since January 2017, his co-leading the team with Pr JM François. 

 

 

General description of the research activity

 

The main focus of our research activity is an integrated approach of the molecular mechanisms by which microorganisms adapt to environmental constraints that are relevant to industrial situation. Our major model study is the yeast Saccharomyces cerevisiae’ on whichis delineated three research topics (i) organization, remodeling, adaptation of cell wall and cell surface using combination of biochemical, molecular and biophysical methods biochemical; (ii) metabolic and genetic regulation of carbon and energy with emphasis on the role of the TPS system in cell physiology, as well as on cell adaptation related to gene expression variability in relation to environmental stress; and (iii) construction or refactoring central carbon metabolism for specific biotechnological objectives such as production of chemical (non-natural) synthons. In addition, our team is collaborating since more about 10 years with the industrial company Adisseo SAS located at INSA on gene discovery and functional genomics programs related to the industrial filamentous fungi Talaromyces versatilis (previously known as Penicillium funiculosum) with a specific focus on expression, regulation and function of glucosylhydrolases/transferases encoding genes. To achieve our goal of ‘integrated physiology, we employed state of the art modern ‘omics’ tools and when requested developed specific analytical (for measuring metabolites, cell wall components) and molecular biology tools (qRT-PCR). Overall, our research activities have a biotechnological-oriented objective that is to propose strategies to optimize the ‘metabolic’ capacity of these microorganisms for objective design, which can be an added-value (chemicals) product in the case of yeast or high expressed/produced glycosyl hydrolase for the fungus. 

 

Key words: yeast, filamentous fungi, functional genomics, cell wall assembly, metabolic regulation, integrated physiology, cell adaptation, synthetic metabolism. 

 

 

Research Topics

Topic 1: Biophysics and molecular biology approaches of the yeast cell wall construction and assembly (PI: Didier Zerbib & Helen Martin-Yken)

  • Structure–function analysis of Knr4 protein, a unique fungal protein implicated in regulation of cell wall assembly
  • Crystallography studies of Knr4 with some of its identified partners (Slt2/Mpk1; Tys1)
  • Search for the mechanism by which Knr4 becomes indispensable in the absence of a functional PKC1-Slt2 (cell wall ) integrity pathway
  • Integrative analysis combining atomic microcopy, biochemical and molecular approaches to apprehend the complexity of the molecular organization of the yeast cell wall

 

Main scientific contributions since 2012

 

  1. Formosa, C., Schiavone, M., Martin-Yken, H., Duval, R.E., François, JM. & Dague, E. (2013). Nanoscale effects of caspofungin on Saccharomyces cerevisiae and Candida albicans.Ant. Agents Chemoth. 57 (8) 3498 -3406.
  2. Pillet, F., Lemonier, S., Schiavone, M., Formosa, C., Martin-Yken, H., François, J & Dague, E (2014). Uncovering by Atomic Force Microscopy of an original circular structure at the yeast cell surface in response to heat shock. BMC Biology, 12:6
  3. Francois, J., Formosa, C., Pillet, F., Schiavone, M., Martin-Yken, M; & Dague, E (2014). Use of Atomic Force Microscopy (AFM) to explore cell wall properties and response to stress in the yeast Saccharomyces cerevisiae Curr Genet, 59: 187 -196.
  4. Schiavone, M., Formosa, C., Martin-Yken, M., Dague, E & François (2014) Combined chemical and enzymatic digestion for a quantitative analysis of the cell wall components in yeasts. FEMS Yeast Res. 14: 933 -947.
  5. Formosa, C., lachaize, V., Galès, C., MP Roles, Martin-Yken, H., François, JM., Duval, RE & Dague, E (2015) Mapping HA-taggeed protein at the surface of living cells by Atomic force microscopy. J. Mol. Recognit., 28 : 1-9.
  6. Schiavone, M., Castex, M., Siekowzki, N., Dague, E & François, J. (2015). Atomic force microscopy allows revealing effects of autolysis processes and differences in nanomechanical properties of industrial yeast strains FEMS Yeast Res., 15, (2) 1-9,
  7. Ran L., Formosa, C., Dagkessamanskaia, A., Dague, E. François, J & Martin-Yken, H. (2015).Combining Atomic Force Micsroscopy and genetics to investigate the role of Knr4 in Saccharomyces cerevisiae sensitivity to K9 killer toxin.Lett. App. NanoBioSc., 4, 4, 306-315
  8. Julien, S., Tondl, P., Durand,F., Dagkessamanskaia,A., van Tilbeurgh, H., Francois,J, Mourey, L., Zerbib, D., Yken-Martin, H. and Maveyraud, L. (2015) Crystallization and preliminary crystallographic studies of the structured core domain of Knr4 from Saccharomyces cerevisiae, Acta Cryst F, F71.
  9. François, J.M (2015) Cell Surface Interference with Plasma Membrane and Transport Processes in Yeasts in Advances in Experimental Medicine and Biology, Volume 892 pp 11-31, doi: 10.1007/978-3-319-25304-6_2. Springer Verlag
  10. Schiavone, M., Elsztein, C., Formosa-Dague, C., Teste, M-A., Martin-Yken, H., Morais, M.A.jr, Dague, E. & François, J. (2016).An Atomic Force Microscopy study of the response of the yeast Saccharomyces cerevisiae to ethanol stress. Appl.Env Microbiol., 82: 4789-4801
  11. Martin-Yken, H., François, J. & Zerbib, D (2016) Knr4: a disordered hub protein at the heart of fungal cell wall signalling. Cellular Microbiology, 18(9) 1217-1227
  12. Teissié, J. and D. Zerbib (2017)." Eradication of Bacteria Via Electropulsation " in Reference Module in Chemistry, Molecular Sciences and Chemical Engineering." Elsevier Inc. Editor-in-Chief, Jan Reedijk Sciencedirect. DOI: 10.1016/B978-0-12-409547-2.13455-9
  13. Teissié, J. and D. Zerbib (2017)." Cell Membrane Electropermeabilization" in Reference Module in Chemistry, Molecular Sciences and Chemical Engineering." Elsevier Inc.  Editor-in-Chief, Jan Reedijk Sciencedirect. DOI: 10.1016/B978-0-12-409547-2.14139-3
  14. Zerbib, D. (2016).  “Bacterial Cell Envelopes: Composition, Architecture, and Origin”. In the Handbook of Electroporation ",. editor, Damijan Miklavcic, publisher, Springer International Publishing  DOI: 10.1007/978-3-319-26779-1_28-1
  15. Schiavone, M., Bringe, K., Martin-Yken, H., François, J M., Castelain, M., Sletmoen, M. & Dague, E (2017)Single molecule dynamic force spectroscopy reveals specific binding of concanavalin A to yeast cell wall mannoproteins. Nanoscale Horizons (submitted)
  16. Schiavone, M., Déjean, S., Sieczkowski, N., Castex, M., Dague, E. and François J M (2017)Integration of biochemical, biophysical and transcriptomics data for investigating the structural and nanomechanical properties of the yeast cell wall. Frontiers in Microbiology, 8: 1809; DOI: 10.3389/fmicb.2017.01806

 

 

 

Topic 2: functional genomics, metabolic regulation and cell adaptation (PI: JL Parrou, CR1-CNRS & JP Capp, MDC-INSA)

 

  • Investigation of the role of the TPS system (Trehalose 6-P synthase/ T6P phosphatase) in yeast energy homeostasis
  • Investigate whether cellular adaptation to stressful conditions may arise in part through an original evolution mechanism, that is dependent upon the stochasticity (or noise) in the expression of key genes.
  • Physiogenomics of Saccharomyces and non-Saccharomyces yeasts and of the industrial filamentous fungus Talaromyces versatilis

 

 

Main scientific contributions since 2012

  1. Hassan H., François, JM & Blanc, P.J. (2012) Effect of amino acids on red pigments and citrinin production in Monascus ruber. J. Food Sci., 77, 156 -159.
  2. Yang, D-D., François, JM & De Billerbeck, G (2012) Cloning and characterization of cDNA encoding an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochate chrysosporium. BMC Microbiology 28;12(1):126
  3. Schlecht, U., Stonge, R., Walther, T., François J, & Davis, RW (2012) Cationic amphiphilic drugs are potent inhibitors of yeast sporulation. Plos One, 7, e42853
  4. Walther, T., Baylac, A., Alkim, C., Vax, A., Cordier, H. & François J (2012) The PGM3 gene encodes the major phosphoribomutase in the yeast Saccharomyces cerevisiae. FEBS Lett., 586: 4114 -4118
  5. Zhang, H., Fauré, R., François, J., Blanc, PJ  De Billerbeck, G. (2013) Xylosylation as an effective means for reducing yeast growth inhibition by 2-phenylethanol. J Basic Microbiol 53, 1 -4
  6. Vidal Espinosa E., De Billerbeck, G., Ardaillon Simoes, D., Schuler, A., François, J & Morais M.A. (2013) Influence of nitrogen supply on the production of higher alcohol/esters and expression of the flavor - related genes in cachaça like-industrial fermentation. Food Chemistry, 138: 701 -708.
  7. Alkim, C., Benbadis, L., Cakar, Z.P. & François, J. (2013) Mechanisms other than activation of iron regulon account for the hyper resistance to cobalt of a yeast strain obtained by evolutionary engineering. Metallomics 5 (8), 1043 - 1060
  8. Walther, T., Mtimet, N., Alkim, C., Vax, A., Loret, M-A., Ullah, AZ., Gancedo, G., Smits, G.J & François J (2013) Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose-6-phosphate dynamics. Biochem.J. 454: 227-237
  9. Rezki, M.A, Benbadis, L., Bendayer, Z., de Billerbeck, G., & François J. (2013) Identification and physiologiocal characterization of indigenous yeasts from Algerian food and dairy products. J.Yeast and Fungal Res., 4: 75 -83.
  10. De la Mare Marion, Guais, O., Bonnin, E., Weber, J. & Francois J (2013) Molecular and biochemical characterization of three GH62 -L-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum. Enz. Microbiol Technol., 51: 351-358
  11. Deng, X., Petitjean, M., Teste, M.A., Trannier, S., François J. & Parrou JL (2014) Similarities and differences in the biochemical and enzymological properties of the four isomaltases from the yeast Saccharomyces cerevisiae. FEBS Open Bio 4, 200-212
  12. Delmas S, Llanos A, Parrou JL, Kokolski M, Pullan ST, Shunburne L & Archer D (2014) Development of an Unmarked Gene Deletion System for the Filamentous Fungi Aspergillus niger and Penicillium funiculosum. Appl Environ Microbiol. Jun;80(11):3484-7.
  13. Walther T., Peyriga L., Alkim C., Liu Y., Agrawal U., Bechter, B., Guo, S., Lardenois, A., Loret, M.O., Martin-Yken, H., Letisse, F., Primig, M. and François J.M (2014). Cell cycle associated metabolic regulation during meiotic development in yeast. BMC Biology, 12: 60
  14. Grondin, E., Cheong Sing, A.S., Caro, Y., DeBillerbeck, G., François, J & Petit, T (2014).Physiological and biochemical characteristics of ethyl-tiglate production pathway in the yeast Geotrichum fragrans. Yeast, DOI: 10.1002/yea.3057
  15. Poueymiro, M., Caalé, AC, François, J., Parrou JL., Peeters, N & Genin, S (2014)A Ralstonia solnacearum type III effector directs the production of the plant metabolites trehalose-6-phosphate. mBio, 5 (6) e062065-14
  16. Vidal, E., Morais, MA, François, J. & deBillerbeck, G (2015) Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: Impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source. Yeast, DOI: 10.10002/yea.3045
  17. Hassan, H., Goma, G & François J. (2015). The influence of carbon/nitrogen ratio on red pigment and citrinin production by filamentous fungi Monascus ruber is dependent on the cultivation mode. Int. Journal Food Science & Technology. DOI: 10.1111/ijfs.12803
  18. LLanos A, François JM, Parrou JL (2015). Tracking the best reference genes for RT-qPCR data normalisation in filamentous fungi. BMC genomics, 16: 71
  19. Liu, J., Martin-Yken, H., Bigey, F., Dequin, S., Francois, J. & Capp, JP (2015)Natural yeast promoter variants exhibit different noise levels conferring distinct selective advantages. Genome Biol Evol., 7 (4) 969 -984.
  20. Petitjean, M., Teste, MA., François, J & Parrou JL (2015).Yeast tolerance to stress relies on Tps1 protein, not on trehalose. J.Biol.Chem., Jun 26;290(26):16177-90; doi: 10.1074/jbc.M115.653899.
  21. Grondin, E., Cheong Sing, A.S., Caro, Y., Raheminadimbny, M., Randrianierenana, A., James, S., Nueno-Palop, C., François J. & Petit, T (2015). A comparative study on the potential of epiphytic yeasts isolated from tropicla fruits to produce flavoring compounds. Int J Food Microbiol., 203, 101 -108.
  22. Liu, J., François, J & Capp, JC (2016)Use of gene expression as experimental parameters to test phenotypic effects. Yeast, 33 209-216
  23. Grondin, E., Shum Cheong Sing, A., Caroa, Y., James, S., Nueno-Palop,C., François, JM.& Petit, T (2017) Flavour production by Saprochaete and Geotrichum yeasts and their close relatives. Food Chem. 237: 677-684.
  24. Petitjean, M., Teste, MA., Leger-Silvestre, I. François, J M. & Parrou, JL (2017). A new function for the yeast trehalose-6-P synthase (Tps1) protein, as key prosurvival factor during growth, chronological ageing and apoptotic stress. Mech. Ageing & Devel. Aug 6. pii: S0047-6374(16)30118-X. doi: 10.1016/j.mad.2016.07.011.
  25. Turanlı-Yıldız, B., Benbadis, L., Alkım, C., Sezgin, T.,  Aksit, A., Gökçe, A., Öztürk,Y., Tarık Baykal, A., Çakar, Z.P. and François J M (2017) In vivo evolutionary engineering for ethanol-tolerance of Saccharomyces cerevisiae haploid cells triggers diploidization. J Biosci. Bioeng. https://doi.org/10.1016/j.jbiosc.2017.04.012
  26. Yang, DD., ZhangJ-J, Rosenzweig, F., De Billerberck, G  & François, J M. (2017) Deciphering the origin, evolutionary fate and the physiological function of the subtelomeric multi-aryl-alcohol dehydrogenases encoded genes family in the yeast Saccharomyces cerevisiae. Applied Env Microbiol (accepted)*
  27. Liu, J., François, JM & Capp, JP (2017) Gene expression noise produces cell-to-cell heterogeneity in eukaryotic homologous recombination rate. Mol Biol Evol (submitted)

 

Patents

  1. François, J., Petitjean, M & Parrou (2016) Yeast with a strong improvement of fermentation capacity. Patent submitted by INSA/Toulouse-Transfer Technologie on February 18th 2016, under n° EP16305188.1

 

 

Topic 3: Synthetic metabolic refactoring and synthetic system machinery (PI: Jean Marie François, PRCE)

 

  • Develop an alternative non-natural-pathway for pentose assimilation which leads to the production of the value-added compounds, such as ethylene glycol and glycolic acid (patent under deposition).
  • Create non-natural metabolic pathways for cost-efficient, sustainable, and petrol-independent production of methionine

 

Main scientific contributions since 2012

 

  1. Schlecht, U., Stonge, R., Walther, T., François J, & Davis, RW (2012) Cationic amphiphilic drugs are potent inhibitors of yeast sporulation. Plos One, 7, e42853
  2. Alkim, C., Benbadis, L., Cakar, Z.P. & François, J. (2013) Mechanisms other than activation of iron regulon account for the hyper resistance to cobalt of a yeast strain obtained by evolutionary engineering. Metallomics 5 (8), 1043 - 1060
  3. Cam, Y., Alkim, C., Trichez, D., Vax, A., Bartolo, F., Besse, Ph., François J & Walther, T (2015) Engineering of a synthetic metabolic pathway for the assimilation of (D)-xylose into added-value chemical. ACS Synthetic Biology, DOI10.1021.acssynbio.5b00103
  4. Alkim, C., Cam, Y., Trichez, D., Auriol, C., Vax, A., Spina, L., Bartolo, F., Besse, P., François, J & Walther, T (2015). Optimisation of ethylene glycol production from (D) xylose via a synthetic pathway implemented in Escherichia coli. Microb. Cell Fact. 14 :127 DOI 10.1186/s12934-015-0312-7
  5. Alkim, C., Trichez, D., Cam, Y., François, J & Walther, T (2016). The synthetic xylulose 1-phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures. BMC Biotechnol. Biofuels, 9:201
  6. Walther Th., Topham, C.M., Irague, R., Auriol, C., Baylac, A., Cordier, H., Dressaire, C., Lozano-Huguet, L., Tarrat, N., Martineau, N., Stodel, M., Malbert, Y., Maestracci, M., Huet, R., André, I., Remaud-Siméon, M & François J M. (2017)Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid. Nature Comm., 8, 15828 doi:10.1038/ncomms15828.
  7. Irague, R., Topham, C., Martineau N, Baylac A , Stodel M., Auriol C., Walther, Th., François Jean M., André I. & Remaud-Siméon M. (2017)Conversion of an aspartate kinase into a malate kinase using a generic HTS assay for kinase screening. ChemCatchem (submitted)

 

 

 

Patents:

  1. Walther, T., François, J M & Huet, R. A novel method of production of 2,4-dihydroxybutyric acid Patent submitted October, 28th 2010 as PCT/FR2010/003153.
  2. Walther, T., François, JM, Huet, R. (2011). A novel method of production of 2,4-dihydroxybutyric. submitted May 25th as PCT/FR2011/000307
  3. Walther, T., Cordiez, H., Topham Ch., Andre I, Remaud-Simeon, M., Huet, R. & François, J. A novel method of production of 2,4-dihydroxybutyric. Submitted October 27th 2011 PCT/FR2011/000578, published May 5th 2012 as WO2012/056318A1
  4. Walther, T., Cordier, H., Dressaire, C. François, J. A production of 2,4-dihydroxybutyric by malyl-CoA pathway. Submitted on April 25th as PCT/FR 2012/001071 and published October 31th as WO2013/160762A2
  5. Walther, T., Dressaire, C., Cordier, H. & François, J. “A method for preparation of 2, 4 dihydroxybutyric acid”. Patent deposition PCT/EP 2013/064619 on 10 July 2013; Priority: brevet US patent July 12th n°61/670,405, published January 16th  2014 as WO2014009435 A1
  6. Walther, T., François, J. "A microorganism modified for the production of 1,3-propanediol". Patent deposition n° PCT/EP 2013/064616 on 10 July 2013, Priority US on July 12th, n° 61/670 389, published January, 16th 2014 as WO2014009432
  7. Senescau, A., François, J., Bernier, M. & Fabre, R (2013) « Biopuce pour la détection et identification de bactéries du genre légionella, kit et procédé d’utilisation ». French patent n° published 21th March 2014 as 2995614
  8. Foncy, J., Trevisiol, E., Severac, C., François, J. (2014) Procédé d’immobilisation d’un composé d’intérêt sur un support selon un motif donné et kit pour sa mise en œuvre. dépôt français n° FR °14 60398 le 29 octobre 2014
  9. Walther, T., Cam, Y & François J (2014) Procédé de production d’au moins un métabolite d’intérêt par transformation d’un pentose dans un microorganisme. Demande de brevet international déposé par INRA n° FR14 61183 du 18 nov 2014 ; Extension n° PCT/FR2015/053138, 18 nov 2015

 

 

Technical resources of the team

The technical resource owned by PHYGE consists of a spore micro dissector (responsible; MA Teste), a flow cytometer (responsible: JP Capp), a HPLC-Dionex coupled to MS and a GC-MS (responsible: L Spina). Other equipment such as centrifuges, mini-autoclave, DNA electrophoresis and PCR machine are shared with other teams. The team has also easy access to Genomic, proteomic, metabolomis/ fluxomics and fermentation platforms hold by skilled engineers at LISBP and TWB

Ongoing Projects and Research collaborations

  1. Project ANR-Grand Emprunt Bioressources & Biotechnologie (2012 -2017) Title : Biologie Synthétique pour la synthèse de molécules chimiques à haute valeur ajoutée à partir de ressources carbonées renouvelables. Acronyme : SYNTHACs.
  2. Project ANR Défi Industriel (2015-2017): Title: Robust engineering and directed evolution of synthetic pathways by integration of genomics and microfluidics. Acronym: SYNPATHIC.
  3. TWB exploratory project (2015 -2016) Title: Robust engineering of synthetic pathways through novel genome design (ROBUST).
  4. TWB exploratory project (2014 -2015). Title: Development of Synthetic Metabolic Pathways for the Assimilation of Pentose Sugars (Pentosys).
  5. TWB exploratory project (2015 -2017). Title: Amélioration de souche vis-à-vis de stress rencontrés en conditions industrielles par une stratégie innovante basée sur la variabilité d'expression des gènes et le tri cellulaire par cytométrie en flux (ROBUSTRAIN)
  6. TWB exploratory project (2017 -2018). Title: Engineering yeast with higher fermentative capacity (Superyeast)
  7. Industrial Support & collaborations with Lallemand Inc,  on Yeast cell wall and yeast fermentation
  8. Industrial support & Collaborations with ADisseo on  Gene discovery in Talaromyces versatilis
  9. Project ANR-ADEME ‘Industrie & agriculture ecoefficientes 2017 -2021 Title: Faisabilité économique de la production de 2, 4 dihydroxybutyric acid par voie biotechnologique. Acronym :  ECOMET-Bio

 

 

 

  • Scientific collaborations with
          

        IBPS-CNRS Toulouse (Dr Laurent Maveyraud & Dr Lionel Mourey)

        LAAS-CNRS Toulouse (Prof C Vieu & Dr Etienne Dague)

        Institute Mathématique Appliquées Toulouse, Group Biostatistic (Dr S Dejean & Prof Ph Besse)

        Université de l’Ile de la Réunion, St Denis (Prof. Thomas Petit)

        TU Delft (Dr Jean Marc Daran & Prof Jack Pronk)

        SupAgro - INRA Montpellier (Dr S Dequin & Prof Bruno Blondin)

        University Pernambuco, Recife, Department of Genetic (Prof Marcos Morais and Prof Diego Simoes)

        University Free Amsterdam (Dr Geertien Smits)                

     

 

               

 

Link to relevant WEB Site