Search

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 β-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 β-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-β-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.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel β-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4–5 A, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL−1 and Vmax 690 μmol·min−1.mg−1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Made available in DSpace on 2019-10-04T12:41:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-04 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazilian Funding Agency: Foundation for Support to Teaching, Research and Assistance - FAEPA of the HCFMRP-USP The zinc finger transcription factor PAC-3/RIM101/PacC has a defined role in the secretion of enzymes and proteins in response to ambient pH, and also contributes to the virulence of species. Herein we evaluated the role of PAC-3 in the regulation of Neurospora crassa genes, in a model that examined the plant-fungi interactions. N. crassa is a model fungal species capable of exhibiting dynamic responses to its environment by employing endophytic or phytopathogenic behavior according to a given circumstance. Since plant growth and productivity are highly affected by pH and phosphorus (P) acquisition, we sought to verify the impact that induction of a Delta pac-3 mutation would have under limited and sufficient Pi availability, while ensuring that the targeted physiological adjustments mimicked ambient pH and nutritional conditions required for efficient fungal growth and development. Our results suggest direct regulatory functions for PAC-3 in cell wall biosynthesis, homeostasis, oxidation-reduction processes, hydrolase activity, transmembrane transport, and modulation of genes associated with fungal virulence. Pi-dependent modulation was observed mainly in genes encoding for transporter proteins or related to cell wall development, thereby advancing the current understanding regarding colonization and adaptation processes in response to challenging environments. We have also provided comprehensive evidence that suggests a role for PAC-3 as a global regulator in plant pathogenic fungi, thus presenting results that have the potential to be applied to various types of microbes, with diverse survival mechanisms. Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Genet, Ribeirao Preto, Brazil Morgana Potrich Coll, Dept Biofunct, Mineiros, Brazil Sao Paulo State Univ, Inst Chem, Dept Biochem & Technol Chem, UNESP, Araraquara, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto, Brazil Sao Paulo State Univ, Inst Chem, Dept Biochem & Technol Chem, UNESP, Araraquara, Brazil FAPESP: 2014/03847-7 FAPESP: 2018/11319-1 FAPESP: 2016/04274-6 CNPq: 305252/2013-5 CNPq: 304222/2013-5 CAPES: 001

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.

Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, β-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (mus) genes in the model fungus Neurospora crassa, i.e. mus-51, mus-52, or mus-53, orthologous to the human genes KU70, KU80, and LIG4, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of mus-52 deletion in N. crassa gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.