Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Characterization of a New Glucose-Tolerant GH1 ß-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity.

Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new ß-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent Tm value was 48.5 °C. The percentages of α-helix (36.3%) and ß-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50-250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC50 = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg-1), pNPG (340.5 ± 18.6 U mg-1), cellobiose (89.3 ± 5.1 U mg-1), and lactose (45.1 ± 0.5 U mg-1), so it had broad specificity. The Vmax values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg-1 toward p-nitrophenyl-ß-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast® 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L-1) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus cellulases already characterized by our research group-CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification.

Corrigendum to analysis of the phosphorylome of Trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

[This corrects the article DOI: 10.1016/j.btre.2021.e00652.].

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

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.

Molecular Characterization of Xyloglucanase cel74a from Trichoderma reesei.

BACKGROUND: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. METHODS: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. RESULTS: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. CONCLUSIONS: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.

Development of novel cocrystal-based active food packaging by a Quality by Design approach.

A way to reduce food waste is related to the increase of the shelf-life of food as a result of improving the package type. An innovative active food packaging material based on cocrystallization of microbiologically active compounds present in essential oils i.e. carvacrol, thymol and cinnamaldehyde was developed following the Quality by Design principles. The selected active components were used to produce antimicrobial plastic films with solidified active ingredients on their surface characterized by antimicrobial properties against four bacterial strains involved in fruit and vegetable spoilage. The developed packaging prototypes exhibited good antimicrobial activity in vitro providing inhibition percentage of 69 (±15)% by contact and inhibition diameters of 32 (±6) mm in the gas phase, along with a prolonged release of the active components. Finally, the prolonged shelf-life of grape samples up to 7 days at room temperature was demonstrated.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

Stepwise Evolution of Molecular Nanoaggregates Inside the Pores of a Highly Flexible Metal-Organic Framework.

The crystalline sponge method (CSM) is primarily used for structural determination by single-crystal X-ray diffraction of a single analyte encapsulated inside a porous MOF. As the host-guest systems often show severe disorder, reliable crystallographic determination is demanding; thus the dynamics of the guest entering and the formation of nanoconfined molecular aggregates has not been in the spotlight. Now, the concept is investigated of the CSM for monitoring the structural evolution of nanoconfined supramolecular aggregates of eugenol guests with displacement of DMF inside the cavities of the flexible MOF, PUM168. The interpretation of the electron density provides a series of unique detailed snapshots depicting the supramolecular guest aggregation, thus showing the tight interplay between the host flexible skeleton and the molecular guests through the DMF-to-eugenol exchange process.

New Genomic Approaches to Enhance Biomass Degradation by the Industrial Fungus Trichoderma reesei.

The filamentous fungi Trichoderma reesei is one of the most well-studied cellulolytic microorganisms. It is the most important fungus for the industrial production of enzymes to biomass deconstruction being widely used in the biotechnology industry, mainly in the production of biofuels. Here, we performed an analytic review of the holocellulolytic system presented by T. reesei as well as the transcriptional and signaling mechanisms involved with holocellulase expression in this fungus. We also discuss new perspectives about control of secretion and cellulase expression based on RNA-seq and functional characterization data of T. reesei growth in different carbon sources, which comprise glucose, cellulose, sophorose, and sugarcane bagasse.

The Duality of the MAPK Signaling Pathway in the Control of Metabolic Processes and Cellulase Production in Trichoderma reesei.

In this study, through global transcriptional analysis by RNA-Sequencing, we identified the main changes in gene expression that occurred in two functional mutants of the MAPK genes tmk1 and tmk2 in Trichoderma reesei during sugarcane bagasse degradation. We found that the proteins encoded by these genes regulated independent processes, sometimes in a cross-talk manner, to modulate gene expression in T. reesei. In the Δtmk2 strain, growth in sugarcane bagasse modulated the expression of genes involved in carbohydrate metabolism, cell growth and development, and G-protein-coupled receptor-mediated cell signaling. On the other hand, deletion of tmk1 led to decreased expression of the major genes for cellulases and xylanases. Furthermore, TMK1 found to be involved in the regulation of the expression of major facilitator superfamily transporters. Our results revealed that the MAPK signaling pathway in T. reesei regulates many important processes that allow the fungus to recognize, transport, and metabolize different carbon sources during plant cell wall degradation.

Characterization of a novel sugar transporter involved in sugarcane bagasse degradation in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known. RESULTS: In our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein-ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), ß-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB). CONCLUSION: The transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.

Cadastro domiciliar

Apresenta a ficha de cadastro domiciliar, seu passo a passo e peculiaridades no preenchimento. A ficha tem como objetivo geral de fazer o cadastramento da família, funcionando como um mecanismo de levantamento populacional e a verificação da abrangência das equipes de saúde.

[A careful look to postmodern tribes: caring for adolescent health in the context of their everyday lives]. / Um olhar sensível às tribos pós-modernas: cuidando da saúde dos adolescentes no cotidiano.

This is a theoretical reflection, based on Michel Maffesoli's Comprehensive Sociology, which is concerned with the health care of adolescents in contemporary everyday life, and particularly with the phenomenon of urban tribes. These are understood as groups of people who have emotional ties, building a bond of sociality towards a common goal. This study focuses on the importance to take into account the lifestyle of adolescents and aims to raise awareness among health professionals about such issues, seeking for strategies tuned with reality and care needs in order to promote health, devising ways to improve caring, rethinking health policies for adolescents in contemporary society.

Um olhar sensível às tribos pós-modernas: cuidando da saúde dos adolescentes no cotidiano / Una mirada sensible a las tribus postmodernas: el cuidado de la salud de los adolescentes en la vida cotidiana / A careful look to postmodern tribes: caring for adolescent health in the context of their everyday lives

Trata-se de uma reflexão teórica, fundamentada na Sociologia Compreensiva de Michel Maffesoli. Tem como objetivo refletir sobre o cuidado com a saúde dos adolescentes no cotidiano contemporâneo, considerando um olhar sensível para o fenômeno das tribos urbanas. As tribos são compreendidas como estruturação de grupos de pessoas que se reúnem afetivamente, construindo um vínculo de socialidade com um objetivo comum. Esta reflexão enfoca a importância de se considerar a maneira de viver dos adolescentes e visa ampliar os horizontes dos profissionais da saúde, em busca de estratégias de atuação, sintonizadas com a realidade e as necessidades de cuidados para promover a saúde, delineando, assim, maneiras de cuidar afetivas e, portanto, mais efetivas, contribuindo para repensar as políticas de atenção à saúde dos adolescentes na contemporaneidade.

Se trata de una reflexión teórica, basada en la Sociología Comprensiva de Michel Maffesoli. Tuvo como objetivo reflexionar sobre el cuidado de la salud de los adolescentes en la vida cotidiana contemporánea, teniendo en cuenta un aspecto sensible en el fenómeno de las tribus urbanas. Estas son entendidas como la estructuración de grupos de personas que se reúnen afectivamente para la construcción de un vínculo de sociabilidad hacia una meta común. Esta reflexión, centrándose en la importancia de considerar el modo de vida de los adolescentes tiene como objetivo ampliar los horizontes de los profesionales de la salud en la búsqueda de estrategias de acción en sintonía con la realidad y necesidades de atención para promover la salud, delineando así formas de cuidar, contribuyendo a repensar las políticas de atención de la salud de los adolescentes en la sociedad contemporánea.

This is a theoretical reflection, based on Michel Maffesoli's Comprehensive Sociology, which is concerned with the health care of adolescents in contemporary everyday life, and particularly with the phenomenon of urban tribes. These are understood as groups of people who have emotional ties, building a bond of sociality towards a common goal. This study focuses on the importance to take into account the lifestyle of adolescents and aims to raise awareness among health professionals about such issues, seeking for strategies tuned with reality and care needs in order to promote health, devising ways to improve caring, rethinking health policies for adolescents in contemporary society.

Ficha de visita domiciliar

Aborda a Ficha de visita domiciliar do e-SUS realizadas pelo agente comunitário de saúde. Sensibiliza sobre a adoção do registro no SISAB/e-SUS, apresenta a diferença entre visita domiciliar e e atendimento no domicílio, apresenta a estrutura e última verão da Ficha, bem como esclarece pontos importantes sobre o preenchimento.