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Analysis of the extracellular proteome from Aspergillus nidulans growing in Avicel, sugarcane bagasse and sugarcane straw

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Emericella Nidulans (strain Fgsc A4 / Atcc 38163 / Cbs 112.46 / Nrrl 194 / M139) (aspergillus Nidulans)

SUBMITTER: Marcelo Rubio

LAB HEAD: André Damásio

PROVIDER: PXD031886 | Pride | 2022-05-09

REPOSITORIES: Pride

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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. In Aspergillus nidulans, 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, 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. The extracelllular proteomes of single deleted mutants growing in Avicel was analysed on a Q-TOF mass spectrometer and revealed an overall reduction in cellulase secretion, and some specifically some changes in the secretion of some enzymes, suggesting an adaptive mechanism adopted to compensate LPMO9s absence during cellulose degradation. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification.

Project description:The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31% of these CAZymes were expressed on cellulose and 29% on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 -glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 -xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo--1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases and other GHs. Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins—GHs, CBMs, transporters, and SBPs—implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.

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:In C4 sugarcane (Saccharum spp. hybrids), photosynthetic activity has been shown to be regulated by the demand for carbon from sink tissues. There is evidence, from other plant species, that sink-limitation of photosynthesis is facilitated by sugar-signaling mechanisms in the leaf that affect photosynthesis through regulation of gene expression. In this work, we manipulated leaf sugar levels by cold-girdling leaves (5oC) for 80 h to examine the mechanisms whereby leaf sugar accumulation affects photosynthetic activity and assess whether signaling mechanisms reported for other species operate in sugarcane. During this time, sucrose and hexose concentrations above the girdle increased by 77% and 81%, respectively. Conversely, leaf photosynthetic activity (A) and electron transport rates (ETR) decreased by 66% and 54%, respectively. Quantitative expression profiling by means of an Affymetrix GeneChip Sugarcane Genome Array was used to identify genes responsive to cold-girdling (56 h). A number of genes (74) involved in primary and secondary metabolic pathways were identified as being differentially expressed. Decreased expression of genes related to photosynthesis and increased expression of genes involved in assimilate partitioning, cell wall synthesis, phosphate metabolism and stress were observed. Furthermore four probe sets homologous to trehalose 6-phosphate phosphatase (TPP; EC 5.3.1.1) and trehalose 6-phosphate synthase (TPS; EC 2.4.1.15) were up- and down-regulated, respectively, indicating a possible role for trehalose 6-phosphate (T6P) as a putative sugar-sensor in sugarcane leaves. Twelve month-old field grown Saccharum spp. (L.) hybrid cv. N19 (N19) cultivated at Mount Edgecombe, KwaZulu-Natal (SASRI) were used in the study, which was conducted in November, 2006. Plants were grown on a 5 x 15 m plot located on a north-east facing slope with a gradient of ca. 10o. Cold-girdles were attached to sugarcane leaves (n=4) for a period of 56 h prior to harvest. The girdle consisted of 0.75 cm (diameter) soft plastic tubing, firmly clamped around each leaf, approximately 30 cm from the leaf base. Cooled water maintained at 5oC was then pumped through the tubing using a Grant LTD6G cooling bath (Grant Instruments, Barrington, Cambridge, UK). At harvest, leaf samples were immediately frozen in liquid nitrogen (–196oC) and subsequently milled in an A11 Basic Analysis Mill (IKA, Staufen, Germany). Ground leaf tissue was stored at –80oC in 50 ml centrifuge tubes prior to analysis.

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Analysis of the extracellular proteome from Aspergillus nidulans growing in Avicel, sugarcane bagasse and sugarcane straw

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