Project description:Background: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant substrates. Brown-rot strain produces carbohydrate-active enzymes involved in the degradation of lignocellulosic materials, along with a non-enzymatic mechanism, via Fenton reaction. Differences in the lignocellulose metabolism occurring even among closely related brown-rots are not completely understood, bringing attention to a multi-omics study of brown-rot L. sulphureus. Results: To evidence the oxidative-hydrolytic mechanism, the Laetiporus sulphureus ATCC 52600 genome was sequenced and the response to lignocellulosic substrates was analyzed by transcriptomics and proteomics. The transcriptomic profile in response to a short cultivation period on in natura sugarcane bagasse revealed 128 out of 12,802 upregulated transcripts. The high upregulated transcripts included a set of redox enzymes along with hemicellulases. The exoproteome in response to extended-time cultivation with Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus grandis revealed 121 proteins. Contrasting to the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 proteins upregulated relative to glucose. The secretome produced on sugarcane bagasse was evaluated in the saccharification of pretreated sugarcane straw by supplementing a commercial cocktail. Additionally, growth analysis revealed that L. sulphureus ATCC 52600 has higher efficiency to assimilate glucose than other mono and disaccharides. Conclusion: This study shows the singularity of L. sulphureus ATCC 52600 relative to other Polyporales brown-rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omic analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.
Project description:Using nylon filter arrays, we analyzed the expression profile of 1536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp.) exposed to the phytormone methyl jasmonate (MeJA) for 0-12 h. 26 ESTs were differentially expressed, including novel genes and also genes that had not previously been reported as being MeJA-inducible. Data are for two independent experiments. Keywords = sugarcane Keywords = methyl jasmonate Keywords = nylon arrays Keywords: time-course
Project description:Using nylon filter arrays, we analyzed the expression profile of 1536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp.) exposed to the phytormone methyl jasmonate (MeJA) for 0-12 h. 26 ESTs were differentially expressed, including novel genes and also genes that had not previously been reported as being MeJA-inducible. Data are for two independent experiments. Keywords = sugarcane Keywords = methyl jasmonate Keywords = nylon arrays
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.