Fungal enzymes to UNLOCK recalcitrant biomass hydrolysis – FUNLOCK

Structural and chemical analyzes of the recalcitrant fraction were performed before and after enzymatic treatment of different substrates in order to identify and overcome the obstacles to efficient hydrolysis. A large panel of markers has been studied in order to follow the progress of enzymatic reactions. The effect of fungal enzyme cocktails on several lignocellulosic residues was evaluated in terms of released sugars and structural modifications of soluble and insoluble fractions. These quantitative and qualitative analyzes were performed using analytical and spectroscopic methods and state-of-the-art biophysical techniques. This approach aimed to identify relevant markers to guide the selection of target enzymes using post-genomic approaches and multivariate analyses. The enzymes were produced heterologously and characterized in depth.

The project allowed identifying and characterizing several fungal strains, including a high-performance basidiomycete strain on the recalcitrant fraction of cellulose that sequentially secretes oxidative and hydrolytic enzymes. From a methodological point of view, a fungal enzyme production protocol was successfully established and allowed to study several lignocellulolytic enzymes in the project. Several markers of deconstruction were followed, allowing a better understanding of the enzymatic hydrolysis step.

The filamentous fungi identified have not yet revealed all their secrets! The study of their enzymatic arsenal will have to be deepened. The identification of deconstruction markers will allow a more detailed study of the enzymatic hydrolysis step to improve the second generation biofuel process from lignocellulosic biomass.

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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.