Project description:The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose- degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran and sugarcane bagasse. The proteins from the fungus extract grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L -1 ), while WBE promoted the higher release of xylose (5.71 g L -1 ). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.
Project description:Transcriptional profiling of A. niger comparing WT strain vs. ΔXlnR strain treated with steam-exploded sugarcane bagasse (SESB) for 6, 12 and 24 h. The main objective was to identifiy genes related to cellulases and hemicellulases, comparing the differences between WT strain and the strain with the disrupted xylanolytic transcriptional activator gene, XlnR, after treatment with steam-exploded sugarcane. The experiment was further validated by real-time PCR, mass spectrometry of secreted proteins and enzymatic assays.
Project description:Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. Analysis of the extracellular proteome (secretome) from Aspergillus nidulans growing in Avicel, sugarcane bagasse and sugarcane straw and analysed by LC-MS/MS in a LTQ Orbitrap Velos revealed that up to five LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are co-secreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that the three main secreted LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose- active enzymes with distinct regioselectivity. Deletion and overexpression studies confirmed that the abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G, less abundant in the secretome, and has an important role by oxidizing crystalline fractions of cellulose. Single or double deletion of these AnLPMO9s partially impair fungal growth on sugarcane straw but not on crystalline cellulose, demonstrating the importance of LPMO9s for the saprophytic fungal lifestyle in the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of other electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification.
Project description:Transcriptional profiling of A. niger comparing WT strain vs. ÎXlnR strain treated with steam-exploded sugarcane bagasse (SESB) for 6, 12 and 24 h. The main objective was to identifiy genes related to cellulases and hemicellulases, comparing the differences between WT strain and the strain with the disrupted xylanolytic transcriptional activator gene, XlnR, after treatment with steam-exploded sugarcane. The experiment was further validated by real-time PCR, mass spectrometry of secreted proteins and enzymatic assays. Three-condition experiment : WT-SESB or ÎXlnR-SESB for 6, 12 and 24 h at 30 oC in batch culture. Firstly, WT and ÎXlnR strains were grown in minimal medium with fructose as carbon source (control), and then transferred to SESB as carbon source.