Filamentous fungi are organisms specialized in the breakdown of plant biomass. This capacity is being exploited for lignocellulosic biomass deconstruction with a view to producing 2nd-generation biofuels.
IFPEN led a research project designed to explore fungal biodiversity and improve the understanding of degradation mechanisms. The project aimed at improving this breakdown process and optimizing its use for industrial purposes. The results demonstrated that adding enzymes produced by Podospora anserina to the Trichoderma reesei cocktail increases the efficiency of the enzyme mixtureproduced: this leads to an increase in the hydrolysis yield of pretreated wheat straw of up to 17%.
An important step in the biochemical production of 2nd-generation biofuels is enzymatic decomposition of lignocellulosic biomass into fermentable sugars. The enzymes are produced by the filamentous fungus Trichoderma reesei that generates a cocktail of cellulases and hemicellulases to break down the plant wall. Although this microorganism is capable of secreting a large quantity of enzymes, the cocktail is not very diversified. With a view to improving the overall activity of the enzyme mixture, a thesis research project explored the potential of these enzymes derived from other fungi to supplement this cocktail.
Working with academic research teams (1) from INRA and the CNRS, IFPEN's biotechnology researchers studied the lignocellulolytic enzymes of the filamentous fungus Podospora anserina via genomic and proteomic analyses. This fungus attracted the researchers' interest as a result of its habitat - it is one of the last to appear on the excrement of ruminants, demonstrating its capacity to feed from recalcitrant substrates - and its arsenal of lignolytic and cellulolytic enzymes, one of the most extensive among all the fungal species sequenced to date.
Following induction of its enzymes by different cellulosic substrates, the secretomes produced led to an increase in the hydrolysis yield of pretreated wheat straw of up to 17% compared to the T. reesei enzyme cocktail alone. Analysis of the composition of the two most effective secretomes revealed the presence of several cellulases and oxidative enzymes acting on cellulose (lytic polysaccharide monooxygenases or LPMO), which could potentially be responsible for this boost effect.
In addition, three cellulases from the same glycoside hydrolase family (GH6) have been biochemically characterized. The researchers demonstrated a different mode of action, consistent with the predictions of the three-dimensional structures(2). In fact, for one of the enzymes, the second loop containing the catalytic site (blue arrow on the figure below) is absent, suggesting an endoglucanase-type activity, which was also confirmed experimentally.
Superimposition of modeled 3D structures of two Podospora anserina cellulases belonging to the GH6 family
The overall results obtained demonstrate the significant potential offered by studies on new enzymes produced by fungal species specialized in the breakdown of recalcitrant plant biomass. This type of research is currently ongoing as part of the ANR "Funlock" project coordinated by the LBCF (Biotechnology of Filamentous Fungi laboratory) at INRA Marseille.
(1) UMR 1163 INRA/University of Aix-Marseille (Biotechnology of Filamentous Fungi laboratory-LBCF) and UMR 6098 CNRS /University of Aix-Marseille (Architecture and Functions of Biological Macromolecules), as part of the ANR E-Tricell project.