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ANR program FUNTUNE  (2015-2019)

Fungi-inspired enzyme cocktails for fine-tuned deconstruction of plant biomass

In this project we explore fungal enzymatic machineries in order to design new fungi-inspired enzyme cocktails able to mitigate recalcitrance of plant biomass from diverse sources. Plant-associated fungi have evolved enzymatic toolboxes to adapt to diverse plant hosts and substrates. These enzymatic toolboxes are the key factors for finely tuned modification of plant cell walls during fungus growth. While wood decayers use a large range of carbohydrate-acting enzymes (CAZymes) and oxidoreductases to degrade plant cell walls, symbiotic and biotrophic pathogenic fungi cause limited and targeted damage to plant cell walls leading to non disruptive cell wall loosening. When switching from biotrophy to destructive necrotrophic growth, hemi-biotrophic plant pathogens secrete specific sets of enzymes that dramatically alter plant cell walls. Our purpose here is to define the sets of fungal enzymes associated with these different levels of plant cell wall deconstruction. By comparative analysis of genomics and transcriptomics data, we analyze the sets of enzymes that are produced simultaneously by fungi to alter plant cell wall structure or integrity. Our goal is to develop optimised combinations of enzymes for the breakdown or transformation of lignocellulosic substrates in vitro. After heterologous expression, purified enzymes and fungi-inspired enzyme combinations are tested for the deconstruction of model lignocellulosic substrates by analysing the extractible fraction of the biomass after enzymatic treatment and by profiling the released molecules. In parallel, we investigate the role of selected sets of enzymes in plant cell wall deconstruction in planta during plant-fungus interactions.

Partners :

- UMR Biodiversité et Biotechnologie Fongique, INRA-Aix Marseille Université, Marseille.

- UMR Interactions Arbres-Microorganismes, INRA-Université de Lorraine, Nancy.

- UMR Biologie et Gestion des Risques En Agriculture, INRA-AgroParisTech, Versailles-Grignon.

- UMR Architecture et Fonction des Macromolecules Biologiques, CNRS-Aix Marseille Université, Marseille.

coordinator: marie-noelle.rosso@univ-amu.fr

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ANR program FUNLOCK (2013-2017)

Fungal enzymes to UNLOCK recalcitrant biomass hydrolysis

The recalcitrance of plant biomass to enzymatic degradation is a multifactorial problem affecting present day industry that can be connected to an incomplete understanding of the relationship between biomass structure and enzymatic performance. The objective of FUNLOCK is to identify novel lignocellulolytic enzymes able to overcome bottlenecks encountered during the enzymatic deconstruction of biomass. Previous exploration of fungal biodiversity allowed the identification of fungal species that potentiate the action of the industrial enzymatic cocktail produced by Trichoderma reesei in the deconstruction of biomass. A variety of complementary approaches can now be used to acquire more comprehensive insights into the enzymatic strategies developed by selected fungi to deconstruct recalcitrant biomass. We propose to perform structural and chemical analysis of this recalcitrant moiety before and after enzymatic treatment to identify obstacles impeding the rapid hydrolysis of the substrate. An enlarged set of markers of deconstruction will be measured to follow the extent of enzymatic action. The action of a variety of fungal secretomes on diverse biomass residues will be evaluated using state of the art analytical and spectroscopic methods to quantify sugar release and biophysical techniques to assess structural modification in the soluble and insoluble fractions, respectively. This comprehensive approach will enable the identification of markers that will guide the selection of enzyme targets from the secretomes using post-genomic approaches and multivariate analysis. Between 50 and 100 enzyme targets will be heterologously produced and characterized by high-throughput approaches. Enzymes of high industrial potential will be selected and studied in detail both fundamentally and in applied setting to assess their effective impact on the substrate and on the saccharification processes. The main expected outcome of the project is the identification and development of novel enzymes and informative process indicators for white biotechnology.

Partners:

INRA BCF Biodiversité et Biotechnologie Fongique

AMU AFMB Architecture et fonction des macromolecules biologiques
INRA BIA BIOPOLYMERES, INTERACTIONS, ASSEMBLAGES
CNRS CERMAV Centre de Recherche sur les Macromolécules Végétales
INRA FARE Fractionnement des Agroressources et Environnement
IFPEN IFP Energies nouvelles

coordinator: jean-guy.berrin@univ-amu.fr

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ANR STOCKACTIF - Programme Bio-Matière & Energies (2012 - 2015)

Active storage of biomass to facilitate its industrial transformation 

The project of "active storage" aims at using the period included between the harvest of the biomass and its use in factory to implement ligninolytic type biological processes allowing facilitation of further biomass deconstruction. All other things being equal, it would mean a kind of piloted silage adapted to the new uses of the biomass, that is keeping its potential of transformation in energy (bioethanol, biogas) or in synthons (by the further fermentation of sugars or by the extraction of phenolic compounds). The earnings could be energetic and environmental at the same time: indeed, in all the ways of using biomass, the phase of reduction of size then pretreatment is a strongly energy-consuming stage (grinding, cooking, explosion with vapor) is generally chemical reactives consuming (acid treatments, alkaline treatments, organosolv treatments,...) which need to be eliminated and which degrade the environmental balance sheet.

Partners
4 INRA joint units  : FARE « Fractionnement des Agro-Ressources et Environnement », Reims -  IATE «Ingénierie des Agropolymères et Technologies Emergentes», Montpellier - BIOEMCO « Biogéochimie et écologie des milieux continentaux », Versailles-Grignon - LBE « Laboratoire de Biotechnologie de l’Environnement », Narbonne
4 industrial partners  : Start-up ENVOLURE « Ecoanalyses Innovantes Haut Débit », Montpellier - SOLAGRO Co, Toulouse - Soufflet Co, Nogent sur Seine - Vivescia Co (Champagne Céréales), Reims.

Contact : Jean-Claude.Sigoillot@univ-amu.fr

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FUTUROL (2008-2017)

 
This project, funded by OSEO Innovation in the frameshift of the SAS Procéthol 2G aims to the development and the commercialisation of a full process of production cellulosic ethanol of 2nd generation coming from a great diversity of lignocellulosic raw materials. The FUTUROL project contains a pilote phase, followed by a prototype phase. Ongoing research in the laboratory fall within pretreatment modules and enzyme production concerned by this research project and aim to :
- Explore the fungal enzymatic biodiversity to identify new enzymes to define new enzymatic cockytails adapted to pretreatment  and saccharification of the differents lignocellulosic substrates (wheatstraw, miscanthus, taillis à courte rotation…)
- Characterise new fungal enzymes for pretreatment and saccharification.

Main partners
Research & Development : INRA, IFP Energies Nouvelles, ARD, Lesaffre
Companies : Total, Tereos, Champagne céréales, ONF.

Contacts
Jean-claude.sigoillot@univ-amu.fr
Jean-Guy.berrin@univ-amu.fr
Isabelle.gimbert@univ-amu.fr
Eric.record@univ-amu.fr

www.projet-futurol.com

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ANR E-Tricel - PNRB ADEME (2008-2012)

Production of 2nd generation biofuels faces the recalcitrance of lignocellulosic substrates and requires large amounts of enzymes that contribute to more than 30% of the price of ethanol to convert biomass into fermentable sugars. Getting increasingly efficient industrial enzyme cocktails is a major objective in this area. The Trichoderma reesei fungus produces reference enzymatic cocktails used in industry, but its genome analysis reveals a limited enzyme potential. In the ANR E-Tricel project, novel approaches have been implemented to identify and test new fungal enzymes able of complementing the secretome of T. reesei. With the screening of natural substrates of hundreds of fungal strains from the CIRM-CF INRA collection (www.cirm.esil.univ-mrs.fr/crbmarseille/), several enzymes of interest were identified, characterized, produced on a large scale and tested under industrial conditions. Among the fungi screened, the most powerful ones improved by more than 20% glucose the yield of conversion of pretreated lignocellulosic biomass.

 Contact
Jean-Guy.berrin@univ-amu.fr

 Partners

- Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB - CNRS, Marseille)

- Institut Français du Pétrole Energies Nouvelles (IFPEN, Rueil-Malmaison)

- Laboratoire Ecologie de la Foret de Guyane (EcoFoG – CIRAD, Kourou) 

- SAFISIS Company (Soustons)