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As we conclude this Special Issue on fungal biology and interactions, it is only appropriate to reflect on the remarkable progress our scientific community has made in unraveling the mysteries of the fungal kingdom [...].
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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.
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Celulase , Glucanos , Hypocreales , Trichoderma , Celobiose/metabolismo , Proteoma/metabolismo , Proteínas de Membrana/metabolismo , Celulose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Celulase/metabolismo , Açúcares/metabolismo , Oligossacarídeos/metabolismo , Trichoderma/metabolismoRESUMO
Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.
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Saccharum , Saccharum/química , Celulose/química , Biotecnologia , Biomassa , Hidrólise , Lignina/químicaRESUMO
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.
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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.
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Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hypocreales/genética , Biomassa , Celulases/genética , Celulases/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólise , Hypocreales/metabolismo , Saccharum/metabolismo , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Fungal diseases have been underestimated worldwide but constitute a substantial threat to several plant and animal species as well as to public health. The increase in the global population has entailed an increase in the demand for agriculture in recent decades. Accordingly, there has been worldwide pressure to find means to improve the quality and productivity of agricultural crops. Antifungal agents have been widely used as an alternative for managing fungal diseases affecting several crops. However, the unregulated use of antifungals can jeopardize public health. Application of fungicides in agriculture should be under strict regulation to ensure the toxicological safety of commercialized foods. This review discusses the use of antifungals in agriculture worldwide, the need to develop new antifungals, and improvement of regulations regarding antifungal use.
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Produtos Agrícolas/crescimento & desenvolvimento , Fungicidas Industriais/farmacologia , Produtos Agrícolas/microbiologia , Controle de Medicamentos e Entorpecentes , Humanos , Doenças das Plantas/prevenção & controle , Saúde PúblicaRESUMO
Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.
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Lignocellulose is a rich and sustainable globally available carbon source and is considered a prominent alternative raw material for producing biofuels and valuable chemical compounds. Enzymatic hydrolysis is one of the crucial steps of lignocellulose degradation. Cellulolytic and hemicellulolytic enzyme mixes produced by different microorganisms including filamentous fungi, yeasts and bacteria, are used to degrade the biomass to liberate monosaccharides and other compounds for fermentation or conversion to value-added products. During biomass pretreatment and degradation, toxic compounds are produced, and undesirable carbon catabolic repression (CCR) can occur. In order to solve this problem, microbial metabolic pathways and transcription factors involved have been investigated along with the application of protein engineering to optimize the biorefinery platform. Engineered Microorganisms have been used to produce specific enzymes to breakdown biomass polymers and metabolize sugars to produce ethanol as well other biochemical compounds. Protein engineering strategies have been used for modifying lignocellulolytic enzymes to overcome enzymatic limitations and improving both their production and functionality. Furthermore, promoters and transcription factors, which are key proteins in this process, are modified to promote microbial gene expression that allows a maximum performance of the hydrolytic enzymes for lignocellulosic degradation. The present review will present a critical discussion and highlight the aspects of the use of microorganisms to convert lignocellulose into value-added bioproduct as well combat the bottlenecks to make the biorefinery platform from lignocellulose attractive to the market.
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Biocombustíveis , Biomassa , Hidrólise , LigninaRESUMO
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.
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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.
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Celulase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Sistema de Sinalização das MAP Quinases , Trichoderma/metabolismo , Celulase/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Saccharum/metabolismo , Trichoderma/genética , Trichoderma/crescimento & desenvolvimentoRESUMO
Trichoderma harzianum species are well known as biocontrol agents against important fungal phytopathogens. Mycoparasitism is one of the strategies used by this fungus in the biocontrol process. In this work, we analyzed the effect of Epl-1 protein, previously described as plant resistance elicitor, in expression modulation of T. harzianum genes involved in mycoparasitism process against phytopathogenic fungi; self cell wall protection and recognition; host hyphae coiling and triggering expression of defense-related genes in beans plants. The results indicated that the absence of Epl-1 protein affects the expression of all mycoparasitism genes analyzed in direct confrontation assays against phytopathogen Sclerotinia sclerotiorum as well as T. harzianum itself; the host mycoparasitic coiling process and expression modulation of plant defense genes showing different pattern compared with wild type strain. These data indicated the involvement T. harzianum Epl-1 in self and host interaction and also recognition of T. harzianum as a symbiotic fungus by the bean plants.
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Parede Celular/metabolismo , Resistência à Doença/genética , Efrina-A1/genética , Efrina-A1/metabolismo , Doenças das Plantas/microbiologia , Trichoderma/fisiologia , Análise por Conglomerados , Biologia Computacional/métodos , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Transporte Proteico , Vesículas Transportadoras/metabolismoRESUMO
BACKGROUND: The signaling second messenger cyclic AMP (cAMP) regulates many aspects of cellular function in all organisms. Previous studies have suggested a role for cAMP in the regulation of gene expression of cellulolytic enzymes in Trichoderma reesei (anamorph of Hypocrea jecorina). METHODS: The effects of cAMP in T. reesei were analyzed through both activity and expression of cellulase, intracellular cAMP level measurement, western blotting, indirect immunofluorescence and confocal microscopy. RESULTS: To elucidate the involvement of cAMP in the cellulase expression, we analyzed the growth of the mutant strain ∆acy1 and its parental strain QM9414 in the presence of the inducers cellulose, cellobiose, lactose, or sophorose, and the repressor glucose. Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner. The expression cel7a, and cel6a genes was higher in the presence of sophorose than in the presence of cellulose, lactose, cellobiose, or glucose. Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources. Concomitantly, our immunofluorescence microscopy and western blot data suggest that in the presence of sophorose, cAMP may regulate secretion of cellulolytic enzymes in T. reesei. CONCLUSIONS: These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei. Finally, our data may help develop new strategies to improve the expression of cel7a and cel6a genes, and therefore, favor their application in several biotechnology fields.