Extracellular vesicles from the mycoparasitic fungus Trichoderma harzianum.

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.

Expression profiling of Clostridium thermocellum B8 during the deconstruction of sugarcane bagasse and straw.

The gram-positive bacterium Clostridium thermocellum contains a set of carbohydrate-active enzymes that can potentially be employed to generate high-value-added products from lignocellulose. In this study, the gene expression profiling of C. thermocellum B8 was provided during growth in the presence of sugarcane bagasse and straw as a carbon source in comparison to growth using microcrystalline cellulose. A total of 625 and 509 genes were up-regulated for growth in the presence of bagasse and straw, respectively. These genes were mainly grouped into carbohydrate-active enzymes (CAZymes), cell motility, chemotaxis, quorum sensing pathway and expression control of glycoside hydrolases. These results show that type of carbon source modulates the gene expression profiling of carbohydrate-active enzymes. In addition, highlight the importance of cell motility, attachment to the substrate and communication in deconstructing complex substrates. This present work may contribute to the development of enzymatic cocktails and industrial strains for biorefineries based on sugarcane residues as feedstock.

Penicillium polonicum a new isolate obtained from Cerrado soil as a source of carbohydrate-active enzymes produced in response to sugarcane bagasse.

Penicillium species have been studied as producers of plant cell wall degrading enzymes to deconstruct agricultural residues and to be applied in industrial processes. Natural environments containing decaying plant matter are ideal places for isolating fungal strains with cellulolytic and xylanolytic activities. In the present study, Cerrado soil samples were used as source of filamentous fungi able to degrade xylan and cellulose. Penicillium was the most abundant genus among the obtained xylan and carboxymethylcellulose degraders. Penicillium polonicum was one of the best enzyme producers in agar-plate assays. In addition, it secretes CMCase, Avicelase, pectinase, mannanase, and xylanase during growth in liquid media containing sugarcane bagasse as carbon source. The highest value for endo-ß-1,4-xylanase activity was obtained after 4 days of growth. Xyl PP, a 20 kDa endo-ß-1,4-xylanase, was purified and partially characterized. The purified enzyme presented the remarkable feature of being resistant to the lignin-derived phenolic compounds, p-coumaric and trans-ferulic acids. This feature calls for its further use in bioprocesses that use lignocellulose as feedstock. Furthermore, future work should explore its structural features which may contribute to the understanding of the relationship between its structure and resistance to phenolic compounds. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03405-x.

Functional screening of a Caatinga goat (Capra hircus) rumen metagenomic library reveals a novel GH3 ß-xylosidase.

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-ß-D-glucopyranoside (pNPG), 4-nitrophenyl-ß-D-xylopyranoside (pNPX) and 4-nitrophenyl-ß-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with ß-glucosidase, ß-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular ß-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 µmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most ß-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, ß-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol.

Transcriptome Profiling-Based Analysis of Carbohydrate-Active Enzymes in Aspergillus terreus Involved in Plant Biomass Degradation.

Given the global abundance of plant biomass residues, potential exists in biorefinery-based applications with lignocellulolytic fungi. Frequently isolated from agricultural cellulosic materials, Aspergillus terreus is a fungus efficient in secretion of commercial enzymes such as cellulases, xylanases and phytases. In the context of biomass saccharification, lignocellulolytic enzyme secretion was analyzed in a strain of A. terreus following liquid culture with sugarcane bagasse (SB) (1% w/v) and soybean hulls (SH) (1% w/v) as sole carbon source, in comparison to glucose (G) (1% w/v). Analysis of the fungal secretome revealed a maximum of 1.017 UI.mL-1 xylanases after growth in minimal medium with SB, and 1.019 UI.mL-1 after incubation with SH as carbon source. The fungal transcriptome was characterized on SB and SH, with gene expression examined in comparison to equivalent growth on G as carbon source. Over 8000 genes were identified, including numerous encoding enzymes and transcription factors involved in the degradation of the plant cell wall, with significant expression modulation according to carbon source. Eighty-nine carbohydrate-active enzyme (CAZyme)-encoding genes were identified following growth on SB, of which 77 were differentially expressed. These comprised 78% glycoside hydrolases, 8% carbohydrate esterases, 2.5% polysaccharide lyases, and 11.5% auxiliary activities. Analysis of the glycoside hydrolase family revealed significant up-regulation for genes encoding 25 different GH family proteins, with predominance for families GH3, 5, 7, 10, and 43. For SH, from a total of 91 CAZyme-encoding genes, 83 were also significantly up-regulated in comparison to G. These comprised 80% glycoside hydrolases, 7% carbohydrate esterases, 5% polysaccharide lyases, 7% auxiliary activities (AA), and 1% glycosyltransferases. Similarly, within the glycoside hydrolases, significant up-regulation was observed for genes encoding 26 different GH family proteins, with predominance again for families GH3, 5, 10, 31, and 43. A. terreus is a promising species for production of enzymes involved in the degradation of plant biomass. Given that this fungus is also able to produce thermophilic enzymes, this first global analysis of the transcriptome following cultivation on lignocellulosic carbon sources offers considerable potential for the application of candidate genes in biorefinery applications.

Bioprospecting Microbial Diversity for Lignin Valorization: Dry and Wet Screening Methods.

Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

Trichoderma/pathogen/plant interaction in pre-harvest food security.

Large losses before crop harvesting are caused by plant pathogens, such as viruses, bacteria, oomycetes, fungi, and nematodes. Among these, fungi are the major cause of losses in agriculture worldwide. Plant pathogens are still controlled through application of agrochemicals, causing human disease and impacting environmental and food security. Biological control provides a safe alternative for the control of fungal plant pathogens, because of the ability of biocontrol agents to establish in the ecosystem. Some Trichoderma spp. are considered potential agents in the control of fungal plant diseases. They can interact directly with roots, increasing plant growth, resistance to diseases, and tolerance to abiotic stress. Furthermore, Trichoderma can directly kill fungal plant pathogens by antibiosis, as well as via mycoparasitism strategies. In this review, we will discuss the interactions between Trichoderma/fungal pathogens/plants during the pre-harvest of crops. In addition, we will highlight how these interactions can influence crop production and food security. Finally, we will describe the future of crop production using antimicrobial peptides, plants carrying pathogen-derived resistance, and plantibodies.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Structural and functional characterisation of xylanase purified from Penicillium chrysogenum produced in response to raw agricultural waste.

Commercial interest in plant cell wall degrading enzymes (PCWDE) is motivated by their potential for energy or bioproduct generation that reduced dependency on non-renewable (fossil-derived) feedstock. Therefore, underlying work analysed the Penicillium chrysogenum isolate for PCWDE production by employing different biomass as a carbon source. Among the produced enzymes, three xylanase isoforms were observed in the culture filtrate containing sugarcane bagasse. Xylanase (PcX1) presenting 35 kDa molecular mass was purified by gel filtration and anion exchange chromatography. Unfolding was probed and analysed using fluorescence, circular dichroism and enzyme assay methods. Secondary structure contents were estimated by circular dichroism 45% α-helix and 10% ß-sheet, consistent with the 3D structure predicted by homology. PcX1 optimally active at pH 5.0 and 30 °C, presenting t1/2 19 h at 30 °C and 6 h at 40 °C. Thermodynamic parameters/melting temperature 51.4 °C confirmed the PcX1 stability at pH 5.0. PcX1 have a higher affinity for oat spelt xylan, KM 1.2 mg·mL-1, in comparison to birchwood xylan KM 29.86 mg·mL-1, activity was inhibited by Cu+2 and activated by Zn+2. PcX1 exhibited significant tolerance for vanillin, trans-ferulic acid, ρ-coumaric acid, syringaldehyde and 4-hydroxybenzoic acid, activity slightly inhibited (17%) by gallic and tannic acid.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 µg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Production of Brazilian human norovirus VLPs and comparison of purification methods.

Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

New dioxygenase from metagenomic library from Brazilian soil: insights into antibiotic resistance and bioremediation.

OBJECTIVES: Putative new dioxygenases were identified in a metagenomic ß-lactam-resistance screening and, given their key role on aromatic metabolism, we raise the hypothesis that these enzymes maybe concomitantly related to antibiotic resistance and aromatic degradation. RESULTS: ORFs of three putative dioxygenases were isolated from resistant metagenomic clones. One of them, CRB2(1), was subcloned into pET24a expression vector and subjected to downstream phenotypic and bioinformatics analyses that demonstrated the "dual effect" of our metagenomic dioxygenase, on antibiotic and aromatic resistance. Furthermore, initial characterization assays strongly suggests that CRB2(1) open-reading frame is an extradiol-dioxygenase, most probably a bicupin domain gentisate 1,2-dioxygenase. This observation is, to our knowledge, the first description of a metagenomic dioxygenase and its action on ß-lactam resistance. CONCLUSION: Unraveling the diversity of antibiotic resistance elements on the environment could not only identify new genes and mechanisms in which bacteria can resist to antibiotics, but also contribute to biotechnology processes, such as in bioremediation.

Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum.

BACKGROUND: The species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum. RESULTS: Data obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction. CONCLUSIONS: The present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Cloning and characterization of a protein elicitor Sm1 gene from Trichoderma harzianum.

A small protein, cysteine-rich, designated SM1, produced by Trichoderma virens and Trichoderma atroviride, acts as elicitor for triggering plant defense reactions. We analyzed Sm1 gene expression of eight different strains of Trichoderma spp. grown on glucose, seeds or roots of beans. Regardless of the carbon source, T37 strain had significantly higher Sm1 expression and was chosen for further studies. When grown on different carbon sources, Sm1 expression was highest on galactose, bean seed, glucose and starch. Sm1 gene from T37 strain was cloned; it had a single exon, and encoded a protein of 138 amino acids, showing high sequence identity with some proteins belonging to the cerato-platanin family.

Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene.

Binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. However, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (PTGS) in the host plants. The aim of this study was to optimize a procedure for expression of norovirus virus-like particles (VLPs) in plants using a binary vector with co-expression of a PTGS suppressor to increase the yield of the target protein. The effects of four plant viral PTGS suppressors on protein expression were evaluated using green fluorescent protein (GFP) as a reporter. Constructs for both GFP and PTGS suppressor genes were co-infiltrated in Nicotiana benthamiana plants, and the accumulation of GFP was evaluated. The most effective PTGS suppressor was the 126K protein of Pepper mild mottle virus. Therefore, this suppressor was selected as the norovirus capsid gene co-expression partner for subsequent studies. The construct containing the major (vp1) and minor capsid (vp2) genes with a 3'UTR produced a greater amount of protein than the construct with the major capsid gene alone. Thus, the vp1-vp2-3'UTR and 126K PTGS suppressor constructs were co-infiltrated at middle scale and VLPs were purified by sucrose gradient centrifugation. Proteins of the expected size, specific to the norovirus capsid antibody, were observed by Western blot. VLPs were observed by transmission electron microscopy. It was concluded that protein expression in a binary vector co-expressed with the 126K PTGS suppressor protein enabled superior expression and assembly of norovirus VLPs.

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses.

BACKGROUND: Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricultural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed, and their insecticidal properties analysed against Spodoptera frugiperda larvae. RESULTS: Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. CONCLUSIONS: Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

New insights in Trichoderma harzianum antagonism of fungal plant pathogens by secreted protein analysis.

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, ß-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.