Dataset Information

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

ABSTRACT: Trichoderma reesei is one of the major producers of holocellulases. It is known that in T. reesei, protein production patterns can change in a carbon source-dependent manner. Here, we performed a phosphorylome analysis of T. reesei grown in the presence of sugarcane bagasse and glucose as carbon source. In presence of sugarcane bagasse, a total of 114 phosphorylated proteins were identified. Phosphoserine and phosphothreonine corresponded to 89.6% of the phosphosites and 10.4% were related to phosphotyrosine. Among the identified proteins, 65% were singly phosphorylated, 19% were doubly phosphorylated, 12% were triply phosphorylated, and 4% displayed even higher phosphorylation. Seventy-five kinases were predicted to phosphorylate the sites identified in this work, and the most frequently predicted serine/threonine kinase was PKC1. Among phosphorylated proteins, four glycosyl hydrolases were predicted to be secreted. Interestingly, Cel7A activity, the most secreted protein, was reduced to approximately 60% after in vitro dephosphorylation, suggesting that phosphorylation might alter Cel7A structure, substrate affinity, and targeting of the substrate to its carbohydrate-binding domain. These results suggest a novel post-translational regulation of Cel7A.

SUBMITTER: Pedersoli WR

PROVIDER: S-EPMC8254082 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

Pedersoli Wellington Ramos WR de Paula Renato Graciano RG Antoniêto Amanda Cristina Campos ACC Carraro Cláudia Batista CB Taveira Iasmin Cartaxo IC Maués David Batista DB Martins Maíra Pompeu MP Ribeiro Liliane Fraga Costa LFC Damasio André Ricardo de Lima ARL Silva-Rocha Rafael R Filho Antônio Rossi AR Silva Roberto N RN

Biotechnology reports (Amsterdam, Netherlands) 20210622

Trichoderma reesei is one of the major producers of holocellulases. It is known that in T. reesei, protein production patterns can change in a carbon source-dependent manner. Here, we performed a phosphorylome analysis of T. reesei grown in the presence of sugarcane bagasse and glucose as carbon source. In presence of sugarcane bagasse, a total of 114 phosphorylated proteins were identified. Phosphoserine and phosphothreonine corresponded to 89.6% of the phosphosites and 10. ...[more]

PMID: 34258241

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Project description:The filamentous fungus Trichoderma reesei is a saprophyte involved with polysaccharides cell wall depolymerization, being the most important industrial source of cellulases, which have been used for bioethanol production. The regulation of cellulase expression is controlled at the transcriptional level and in a carbon source-dependent manner. However, the signaling pathways and mechanisms involved in regulating the expression of these enzymes in T. reesei are still poorly understood. In this study, we demonstrate through transcriptional analysis by RNA-Seq the main changes in gene expression that occur in functional mutants for two genes of the MAPK, tmk1 and tmk2. The results obtained allowed us to identify that these proteins in T. reesei regulate independent processes, but sometimes might regulate the same process through different mechanisms of action, being responsible for modulating gene expression in this organism. Regarding to Δtmk2 strain, it was demonstrated that growth in sugarcane bagasse and glucose modulates the expression of genes involved with chromatin remodeling, metabolism of carbohydrates and cell signaling genes such as GPCR, calcium signaling and phospholipases. On the other hand, deletion of MAPK TMK1 promotes more discrete changes in the transcriptional profile and the main changes are related to the decrease in the expression of the major genes for cellulases and xylanases, repressing the expression of transporters belonging to the MFS family, transporters of amino acids and ions such as Ca2+ and Mg2+. Our results revealed that the MAPK signaling pathway in T. reesei regulates many important processes that allow the fungus to promote the recognition, transport and metabolism of different carbon sources during the process of cell wall degradation. Therefore, the clustering of these data will contribute to the construction of a global model for the events that occur during the degradation of lignocellulose in T. reesei, which will lead to the development of more efficient strains in the plant biomass degradation.

Project description:BackgroundSecond generation (2G) ethanol is produced by breaking down lignocellulosic biomass into fermentable sugars. In Brazil, sugarcane bagasse has been proposed as the lignocellulosic residue for this biofuel production. The enzymatic cocktails for the degradation of biomass-derived polysaccharides are mostly produced by fungi, such as Aspergillus niger and Trichoderma reesei. However, it is not yet fully understood how these microorganisms degrade plant biomass. In order to identify transcriptomic changes during steam-exploded bagasse (SEB) breakdown, we conducted a RNA-seq comparative transcriptome profiling of both fungi growing on SEB as carbon source.ResultsParticular attention was focused on CAZymes, sugar transporters, transcription factors (TFs) and other proteins related to lignocellulose degradation. Although genes coding for the main enzymes involved in biomass deconstruction were expressed by both fungal strains since the beginning of the growth in SEB, significant differences were found in their expression profiles. The expression of these enzymes is mainly regulated at the transcription level, and A. niger and T. reesei also showed differences in TFs content and in their expression. Several sugar transporters that were induced in both fungal strains could be new players on biomass degradation besides their role in sugar uptake. Interestingly, our findings revealed that in both strains several genes that code for proteins of unknown function and pro-oxidant, antioxidant, and detoxification enzymes were induced during growth in SEB as carbon source, but their specific roles on lignocellulose degradation remain to be elucidated.ConclusionsThis is the first report of a time-course experiment monitoring the degradation of pretreated bagasse by two important fungi using the RNA-seq technology. It was possible to identify a set of genes that might be applied in several biotechnology fields. The data suggest that these two microorganisms employ different strategies for biomass breakdown. This knowledge can be exploited for the rational design of enzymatic cocktails and 2G ethanol production improvement.

Project description:The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol.

Project description:BackgroundOur dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.ResultsAnalyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.ConclusionOur work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.

Project description:BackgroundOn-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.ResultsThe effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.ConclusionsThe sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.

Dataset Information

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

Publications

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

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