RESUMEN
Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.
Asunto(s)
Cationes Bivalentes/química , Proteínas Fúngicas/aislamiento & purificación , Glicósido Hidrolasas/aislamiento & purificación , Hypocreales/enzimología , Familia de Multigenes , Secuencia de Aminoácidos , Secuencia de Bases , Biodegradación Ambiental , Simulación por Computador , Secuencia de Consenso , Minería de Datos , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/genética , Modelos Moleculares , Filogenia , Polisacáridos/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Azúcares/metabolismo , TemperaturaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMEN
Understanding relationships between genes responsible for enzymatic hydrolysis of cellulose and synergistic reactions is fundamental for improving biomass biodegradation technologies. To reveal synergistic reactions, the transcriptome, exoproteome, and enzymatic activities of extracts from Trichoderma harzianum, Trichoderma reesei and Trichoderma atroviride under biodegradation conditions were examined. This work revealed co-regulatory networks across carbohydrate-active enzyme (CAZy) genes and secreted proteins in extracts. A set of 80 proteins and respective genes that might correspond to a common system for biodegradation from the studied species were evaluated to elucidate new co-regulated genes. Differences such as one unique base pair between fungal genomes might influence enzyme-substrate binding sites and alter fungal gene expression responses, explaining the enzymatic activities specific to each species observed in the corresponding extracts. These differences are also responsible for the different architectures observed in the co-expression networks.
Asunto(s)
Enzimas/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Proteómica/métodos , Trichoderma/fisiología , Productos Biológicos/análisis , Biomasa , Celulosa/química , Enzimas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Hidrólisis , Filogenia , Análisis de Secuencia de ARN/métodosRESUMEN
BACKGROUND: Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. RESULTS: In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. CONCLUSIONS: Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.
Asunto(s)
Biocombustibles , Metabolismo de los Hidratos de Carbono , Biología Computacional , Trichoderma/enzimología , Celulosa/metabolismo , Perfilación de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Anotación de Secuencia Molecular , Polisacáridos/metabolismoRESUMEN
Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Choque Térmico Pequeñas/química , Proteínas de Choque Térmico/química , Xylella/química , Secuencias de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequeñas/metabolismo , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Xylella/metabolismoRESUMEN
Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.
Asunto(s)
Trichoderma/genética , Trichoderma/metabolismo , Biodegradación Ambiental , Biocombustibles , Biomasa , Biotecnología , Celulasa/genética , Celulasa/metabolismo , Cromosomas Artificiales Bacterianos/genética , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Genoma Fúngico , HidrólisisRESUMEN
Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigellosis, which mainly affects children and the elderly in both underdeveloped and developed countries. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Among these proteins is the translocator IpaC, which plays an important role in the invasion process; IpaC is implicated in pore formation in the host cell membrane and induces cytoskeletal rearrangements in macrophages and epithelial cells, thereby promoting bacterial entry. The ability of IpaC to insert onto the plasma membrane is due to a large nonpolar region of the protein structure. This characteristic also renders difficulties in recovery and purification when the protein is expressed in E. coli. Several works have considered different methodologies for the improved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the induction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches.