Trichoderma Reesei Research Articles

Variation of taste and odor compounds in tea beverage after microbial fermentation by HPLC–MS, GC×GC–O–MS, GC–MS, and sensory evaluation

Tea beverages are widely welcomed by consumers, and their flavor quality is particularly important. In this study, different tea extracts were fermented by Trichoderma reesei (TR), Aspergillus niger (AN), and lactic acid bacteria (LAB) to obtain the tea beverages, which were characterized by sensory evaluation as well as instrumental analysis. Nonvolatile compounds in tea beverages were determined by high-performance liquid chromatography–mass spectrometry (HPLC–MS), and volatile compounds were determined by solid-phase microextraction (SPME) combined with comprehensive two-dimensional gas chromatography–olfactory–mass spectrometry (GC×GC–O–MS) and gas chromatography–mass spectrometry (GC–MS). The data were analyzed by both principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The bitter and astringent galloylated catechins in tea beverages were hydrolyzed after TR and AN fermentation. Then after fermentation with LAB, some organic acid and phenolic compounds were decarboxylated, and some saccharides were also metabolized. Meanwhile, the increased content of alcohols and ketones in tea beverages affected the overall aroma. These results demonstrate the positive effects of microbial fermentation on the processing of tea beverages and provide technique support for comprehensive flavor quality control.

Extraction of Cellulases Produced through Solid-State Fermentation by Trichoderma reesei CCT-2768 Using Green Coconut Fibers Pretreated by Steam Explosion Combined with Alkali

The industrial processing of coconut to produce valuable foods, such as water and milk, generates large volumes of waste, especially the fruit shell. Despite this, material can be used in bioprocess applications, e.g., the production of enzymes, its recalcitrance hinders the cultivation of microorganisms, and low productivity is usually achieved. In this study, the production of cellulolytic enzymes through solid-state fermentation (SSF) and their extraction was investigated using the green coconut fiber pretreated by steam explosion, followed by alkali. The fungus Trichoderma reesei CCT-2768 was cultivated, using an experimental design, to study the effect of the water activity and the amount of biomass in the reactor. The combination of the pretreatment strategies yielded more porous biomass, with less hemicellulose (5.38%, compared to 10.15% of the raw biomass) and more cellulose (47.77% and 33.96% in the pretreated and raw biomasses, respectively). The water activity significantly affected the production of cellulases, with maximum activity yielded at the highest investigated value (0.995). Lastly, the extraction of the enzymes from the cultivation medium was studied, and a 9 g/L NaCl solution recovered the highest CMCase and FPase activities (5.19 and 1.19 U/g, respectively). This study provides an important contribution to the valorization of the coconut residue through (i) the application of the steam explosion technology to optimize the production of cellulases using the SSF technology and (ii) their extraction using different solvents.

Optimization of culture conditions for xylanase production by mixed fungal fermentation: Effects on pretreated maize cobs

The use of microorganisms for xylanase production plays important role in the bioconversion of lignocelluloses and also required in huge amount for industrial level application. This necessitates the need to select potent microorganisms for xylanase production, followed by optimization of media components for enhanced production. The effects of altering cultural fermentation conditions on the xylanase production ability in maize cobs were investigated. A consortium of four fungi; Lenzites betulina, Trichoderma reesei, Lachnocladium specie and Aspergillus niger were used to carry out single and mixed solid-state fermentation on NaOH pretreated maize cobs. Optimization of fermentation factors were carried out from ten groups of individual and co-fermented fungal combinations. L. flavidum was found to be the most effective xylanase producer with optimal conditions at pH of 5.5, moisture 75%, inoculum concentration at 5-6 x 103 spores/ml, incubation period of 7-9 days and 1% peptone as the best nitrogen media supplement. Variation to different degrees in the degradation of the maize cobs were observed. A 10% decrease in cellulose was observed with co- cultures of T. reesei and A. niger and a 15% decrease in the hemicellulose fraction. The biotechnological potential of corn cobs has been enhanced by the screening and optimizing the culture conditions.

Predicting mechanical properties of self-healing concrete with Trichoderma Reesei Fungus using machine learning

Predicting mechanical properties of self-healing concrete with Trichoderma Reesei Fungus using machine learning

Enhancing the Production Performance and Nutrient Utilization of Laying Hens by Augmenting Energy, Phosphorous and Calcium Deficient Diets with Fungal Phytase (Trichoderma reesei) Supplementation

A ten-week trial was conducted to evaluate the enhancement of production performance and nutrient utilization of laying hens through augmenting energy, phosphorous, and calcium deficient diets with fungal phytase (Trichoderma reesei) supplementation. 720 Hy-line Brown hens aged 28 weeks were randomly divided into 5 groups; each group had 8 replicates of 18 hens. Five experimental diets were prepared and fed to corresponding groups. A positive control (PC) diet contained 3.50% of calcium (Ca), 0.32% of non-phytate phosphorus (NPP), and apparent metabolic energy (AME) of 11.29MJ/kg, while a negative control (NC) diet contained 3.30% of Ca, 0.12% of NPP, and lower AME of 300 kJ/kg. The other three diets were supplemented with 250 FTU/kg phytase (PHY-250), 1000 FTU/kg phytase (PHY-1000), and 2000 FTU/kg phytase (PHY-2000) in addition to a regular NC diet. Results indicated that the positive control (PC) diet group had higher body weight gain, egg weight, and average daily feed intake. However, laying rate, egg mass, and FCR were most improved in the PHY-2000 group, followed by the PHY-1000 and PHY-250 groups (p < 0.05). Improved yolk color was most notable in laying hens fed the diet with PHY-1000 as opposed to the PC and NC groups (p < 0.05), but no overall difference was found among all of the phytase treated groups. The apparent availability of dry matter, energy, phosphorus, and phytate P was significantly higher in the PHY-2000 group than in the PC and NC groups (p < 0.05). Compared to the PC group, nitrogen retention was significantly higher in the PHY-1000 group, while calcium availability was higher in the PHY-250 group. The results suggested that the addition of phytase to diets with low P, Ca, and AME improved laying performance and apparent availability of dietary nutrients. Thus, it was concluded that the laying hen diet could be supplemented with 1000–2000 FTU/kg phytase for improving laying production and nutrient availability and mitigating the negative impact of reduced nutrient density in laying hen diets.

Safety evaluation of an extension of use of the food enzyme containing cellulase, endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase from the non-genetically modified Trichoderma reesei strain AR-256.

The food enzyme containing cellulase (EC 3.2.1.4), endo-1,3(4)-β-glucanase (EC 3.2.1.6) and endo-1,4-β-xylanase (EC 3.2.1.8) is produced with the non-genetically modified Trichoderma reesei strain AR-256 by AB-Enzymes GmbH. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in seven food manufacturing processes. Subsequently, the applicant requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of nine food manufacturing processes: processing of cereals and other grains for the production of 1) baked products, 2) cereal-based products other than baked, 3) brewed products, 4) starch and gluten fractions, 5) distilled alcohol; processing of fruits and vegetables for the production of 6) wine and wine vinegar, 7) juices, 8) fruit and vegetable products other than juices and 9) fruit-derived distilled alcoholic beverages other than from grape. As the food enzyme-total organic solids (TOS) is removed from or not carried into the final foods in three food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining six processes. It was up to 4.049 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level (NOAEL) reported in the previous opinion (939 mg TOS/kg bw per day), the Panel derived a revised margin of exposure of at least 232. Based on the revised exposure calculation and the outcome of the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Pengaruh Rasio Crude Enzim Selulase Trichoderma reesei dan Aspergillus niger terhadap Kadar Gula dan Bioetanol Fermentasi Kulit Kacang Tanah (Arachis hypogaea L.) Menggunakan Zymomonas mobilis

The need for fuel oil in Indonesia is increasing along with the increase in the number of people's consumption in the fields of transportation, industry, and economy. While the source of petroleum fuel in Indonesia is running low. Therefore, it is necessary to utilize alternative energy, namely bioethanol. Bioethanol can be produced from organic waste that contains high cellulose, one of which is peanut shells. This study aims to determine the effect of the ratio of crude cellulase enzyme Trichoderma reesei and Aspergillus niger on sugar and ethanol content of peanut shell fermentation using Zymomonas mobilis, and to determine the ratio of crude cellulase enzyme that produces the highest sugar and ethanol content of peanut shell fermentation using Z. mobilis. This research is an experimental study with independent variables, namely the ratio of crude cellulase enzyme T. reesei : A. niger (0:0), (1:0), (0:1), (1:1), (1:2), (2:1), (1:3), and (3:1) and the dependent variable of sugar and biethanol content. Data analysis of experimental research results using ANOVA and DMRT test. The addition of crude cellulase enzyme from T. reesei and A. niger affects the sugar content and bioethanol from peanut shell fermentation. The ratio of crude cellulase enzyme from T. reesei and A. niger that produces the highest sugar content is in the treatment P5 ratio of crude enzyme T. reesei: A. niger (2:1) which is 0.14 g/mL and the highest ethanol content from groundnut shell fermentation is in the treatment of P8 crude enzyme ratio of T. reesei : A. niger (1:3) which is 2.23%.

Conditions for CaCO3 Biomineralization by Trichoderma Reesei with the Perspective of Developing Fungi-Mediated Self-Healing Concrete.

Concrete, a widely used building material, often suffers from cracks that lead to corrosion and degradation. A promising solution to enhance its durability is the use of fungi as self-healing agents, specifically by harnessing their ability to promote calcium carbonate (CaCO3) precipitation on their cell walls. However, the ideal conditions for CaCO3 precipitation by the filamentous fungal species Trichoderma reesei are still unclear. In this study, the biomineralization properties of T. reesei in liquid media are investigated. Two different calcium sources, calcium chloride (CaCl2) and calcium lactate are tested, at varying concentrations and in the presence of different nutritional sources that support growth of T. reesei. This study also explores the effects on fungal growth upon adding cement to the medium. Calcium lactate promotes greater fungal biomass production, although less crystals are formed as compared to samples with CaCl2. An increasing calcium concentration positively influences fungal growth and precipitation, but this effect is hindered upon the addition of cement. The highest amounts of biomass and calcium carbonate precipitation are achieved with potato dextrose broth as a nutritional source. By identifying the optimal conditions for CaCO3 precipitation by T. reesei, this study highlights its potential as a self-healing agent in concrete.

Isolation and Molecular Screening of Fungus as Agents in Cellulolytic Transformation Materials from Symbiotic Lichen

The abundance of lignocellulosic waste makes it a potential source for advanced biomaterials through various transformation processes. Lignocellulosic biomass transformation to advanced biomaterials involves enzymes from extracellular metabolites of microorganisms capable of hydrolyzing lignocellulose. This research was to molecularly screen fungi found in symbiotes of lichen endemic to trees growing in North Minahasa (North Sulawesi) with lignocellulolytic transformation enzymes. Molecular screening was conducted from identified fungi isolates based on partial genetical analysis on the locus of Internal Transcribed Spacer (ITS) of the fungi’s ribosomal DNA. Fungi isolates screening identified Trichoderma koningiopsis (isolate HZA8 and isolate HZA6), Penicillium sumatraense (strain CBS 127365 and strain CBS 130380), Trichoderma hamatum (isolate PAN12-45 and isolate PAN12-05), Aspergillus aculeatus (strain A1.9 18S), Aspergillus aculeatus (isolate XSD-74), Trichoderma reesei (strain S2606 and isolate 5A14). Molecular identification and BLAST homology of potentially lignocellulolytic fungi isolates rDNA indicated that isolate KB2 had close relationship with Trichoderma reesei at 100% degree of closeness and an index of cellulolytic activity of 1.19. While isolate KB3 appeared closely related to Aspergillus aculeatus at 99.83% degree of closeness and an index of cellulolytic activity of 1.57. Therefore, the potential of developing bioprocess industries in general and in particular is most probable.

First record of Fusarium brachygibbosum as a causal agent of seed decay and damping-off disease on cotton in Iraq and Control using some bioagents

The results of the isolation and identification of pathogens accompanying the symptomatic seeds and seedlings of cotton showed that the most common fungus was Fusarium brachygibbosum. Molecular identification of the studied fungus was performed using the universal primers: the results of the genetic analysis revealed the identities of the fungus as follows: a 100% identity for F. brachygibbosum that was deposited at the GenBank under accession number ON738702.1. This fungus has shown high pathogenicity against cotton seeds and seedlings by severely reducing their Germination and growth and treating cotton seeds with the biological factors of Trichoderma spp. It revealed a high efficiency in reducing disease incidence and increasing cotton germination percentage. Trichoderma viride showed the highest ability to increase seed germination to 94.44%. In comparison, the lowest ability reached 77.77 % in Trichoderma pseudokoningii and Trichoderma reesei—the results of extracting toxins from the filters of Trichoderma spp. The study's use of trichodermin and Gliotoxin showed the presence of trichodermin and Gliotoxin in large quantities. The percentage of toxin inhibition was significant against the growth of pathogenic fungi. The highest percentage of inhibition was 86.1% for the isolate Trichoderma koningiopsis, and the lowest percentage was 66.65% for the isolate Trichoderma reesei. As for the effect of isolates of resistant fungus on the Pathogen in the field, the highest germination rate was 100%, and the inhibition rate was 0.00% when using the biological preparation prepared from the isolates (T. viride, T. pseudokoningii, T. koningiopsis and T. reesei). Keywords: Fusarium brachygibbosum; Trichoderma spp.; Trichodermin; gliotoxin; Biological control.

Evaluation of the Efficacy of Trichoderma species and their Fungal Toxins in the Eradication of Alternaria alternata Causing Seeds Decay and Damping-off Disease on Cotton in Iraq

This study aimed to isolate and identify the pathogens accompanying the rotting and death of cotton seeds and seedlings. Also, some Trichoderma spp. were assessed against the fungal pathogen associated with the disease. The results showed that one of the essential isolated fungi, Alternaria alternata, has demonstrated high virulence in attacking cotton seeds and seedlings and reducing germination and growth. This fungus was identified based on its morphological and molecular characteristics. The Trichoderma species applied have shown high efficiency in reducing infection rates and increasing cotton germination percentage. Every isolate of Trichoderma showed a high efficiency against the fungus A. alternata by providing the highest antagonistic ability, reaching 93.75%. The highest percentage of inhibition growth of the pathogen (86.11%) was achieved by Trichoderma koningiopsis, while the lowest percentage of inhibition growth of the pathogen was 66.65 % for Trichoderma reesei. However, the biological formula prepared from species Trichoderma viride, Trichoderma pseudokoningii, Trichoderma koningiopsis and Trichoderma reesei displayed the highest percentage of inhibition of 100% against the fungus A. alternata. Keywords. Alternaria alternata; Trichoderma spp.; Trichodermin; gliotoxin; Biological control.

Enzymatic Valorization of Lignocellulosic Biomass—The Influence of Deep Eutectic Solvents and Ionic Liquids on the Activity of Cellulolytic Enzymes

In recent years, there has been a steady increase in the interest in changing lignocellulose processing technologies from harmful and nonecological to more ecological and sustainable. Deep eutectic solvents (DESs) and ionic liquids (ILs) are green, efficient solvents/catalysts for biomass pretreatment as well as its further transformation. In many cases, they are used together with biocatalysts. Additionally, in processes where DESs and ILs are used for the pretreatment of biomass, before enzymatic transformation, traces of these solvents can influence the activity of biocatalysts. For this reason, it is important to evaluate the effect of novel, green solvents on the activity of enzymes widely applied in the biomass valorization processes. In this work, we present the impact of chosen DESs and ILs, most often applied in biomass pretreatment and transformation, on the enzymatic activity of popular cellulolytic enzymes and enzymatic preparations (namely, cellulases from Aspergillus niger, Trichoderma reesei, and Viscozyme L). The enzymatic activity was investigated in the process of transformation of cellulose or biomass to glucose. The results showed that many of the solvents tested had a negative effect on enzymatic activity, suggesting a separation of pretreatment and enzymatic processes.

Overexpression of the Transcription Factor Azf1 Reveals Novel Regulatory Functions and Impacts β-Glucosidase Production in Trichoderma reesei.

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the β-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these β-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes.

The Preparation and Properties of Recombinant Forms of GH74 Family Xyloglucanase from the Trichoderma reesei Fungus

The Preparation and Properties of Recombinant Forms of GH74 Family Xyloglucanase from the Trichoderma reesei Fungus

Structure-guided engineering of transcriptional activator XYR1 for inducer-free production of lignocellulolytic enzymes in Trichoderma reesei

The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry. XYR1 is the major transcriptional activator of cellulases and hemicellulases in T. reesei. However, rational engineering of XYR1 for improved lignocellulolytic enzymes production has been limited by the lack of structure information. Here, alanine 873 was identified as a new potential target for the engineering of XYR1 based on its structure predicted by AlphaFold2. The mutation of this residue to tyrosine enabled significantly enhanced production of xylanolytic enzymes in the medium with cellulose as the carbon source. Moreover, xylanase and cellulase production increased by 56.7- and 3.3-fold, respectively, when glucose was used as the sole carbon source. Under both conditions, the improvements of lignocellulolytic enzyme production were higher than those in the previously reported V821F mutant. With the enriched hemicellulases and cellulases, the crude enzymes secreted by the A873Y mutant strain produced 51 % more glucose and 52 % more xylose from pretreated corn stover than those of the parent strain. The results provide a novel strategy for engineering the lignocellulolytic enzyme-producing capacity of T. reesei, and would be helpful for understanding the molecular mechanisms of XYR1 regulation.

Genome-wide prediction and transcriptome analysis of sugar transporters in four ascomycete fungi

The import of plant-derived small sugars by sugar transporters (STs) has received increasing interest due to its important biological role and great industrial potential. STs are important targets of genetic engineering to improve fungal plant biomass conversion. Comparatively analysis of the genome-wide prevalence and transcriptomics of STs was performed in four filamentous fungi: Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens and Trichoderma reesei. Using phylogenetic analysis and literature mining, their predicted STs were divided into ten subfamilies with putative sugar specificities assigned. In addition, transcriptome analysis revealed complex expression profiles among different STs subfamilies and fungal species, indicating a sophisticated transcriptome regulation and functional diversity of fungal STs. Several STs showed strong co-expression with other genes involved in sugar utilization, encoding CAZymes and sugar catabolic enzymes. This study provides new insights into the diversity of STs at the genomic/transcriptomic level, facilitating their biochemical characterization and metabolic engineering.

Mono- and Dimeric Sorbicillinoid Inhibitors Targeting IL-6 and IL-1β from the Mangrove-Derived Fungus Trichoderma reesei BGRg-3.

Four new sorbicillinoids, named trichodermolide E (1), trichosorbicillin J (2), bisorbicillinolide B (3), and demethylsorbiquinol (5), together with eight known compounds (4, 6-12), were isolated from the cultures of the mangrove-derived fungus Trichoderma reesei BGRg-3. The structures of the new compounds were determined by analyzing their detailed spectroscopic data, while the absolute configurations were further determined through electronic circular dichroism calculations. Snatzke's method was additionally used to determine the absolute configurations of the diol moiety in 1. In a bioassay, compounds 7 and 10 performed greater inhibitory activities on interleukin-6 and interleukin-1β than the positive control (dexamethasone) at the concentration of 25 μM. Meanwhile, compounds 5 and 6 showed potent effects with stronger inhibition than dexamethasone on IL-1β at the same concentration.

341 Effect of Multispecies Fungal Culture Extract Supplementation on the Growth Performance and Carcass Characteristics of Feedlot Cattle

Abstract To evaluate the effect of supplementing feedlot cattle with a multispecies fungal culture extract (MFE; Aspergillus flavus, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Phlebia radiata, Pyricularia oryzae, Stropharia coronilla, Thanatephorus cucumeris, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride) in growth performance and carcass characteristics, Angus × SimAngus-crossbred steers (n = 14) and heifers [n = 46; body weight (BW) 344 ± 21 kg] were used in randomized complete block design. Cattle were blocked by BW and housed in 4 feedlot pens (randomly assigned 2 pens per treatment) where a hay-based diet was fed for 84 d (backgrounding), followed by a ground corn-based finishing diet. Subsequently, cattle were harvested upon reaching desired 12th rib fat thickness (2.0 cm). Diets were offered ad libitum and with (MFE) or without (CON) the inclusion of the multispecies fungal culture extract supplement. Steers were weighed at 28-d intervals throughout the experimental period and carcass data were provided by a USDA grader. All data were analyzed using the PROC MIXED procedure of SAS considering the fixed effect of treatment and sex, and the random effect of block and pen (block). During the 84-d period of the backgrounding phase, feeding MFE increased (P = 0.05, Table 1) the average daily gain (ADG) by ~10%, and tended to increase (P = 0.06, Table 1) the dry matter intake (DMI). No differences were observed in the gain to feed ratio (G:F) between treatments (P = 0.67, Table 1). At the conclusion of the 84-d period, cattle fed MFE were heavier (P = 0.04, Table 1) than cattle in the CON treatment. In the finishing phase, no treatment differences were observed (P ≥ 0.36) for the ADG, DMI, and G:F. At the end of the experiment, cattle supplemented with MFE were heavier (P = 0.04, Table 1) than cattle in the CON treatment. The quantity of days on feed in the finishing diet required to reach harvesting target was not different (P = 0.60) among treatments. For the carcass characteristics, dietary inclusion of the MFE tended to increase the hot carcass weight (P = 0.10, Table 2) and increased the marbling score (P = 0.01, Table 2). No treatment effect was detected (P ≥ 0.25, Table 2) for the dressing, longissimus muscle area, 12th rib fat thickness, kidney-pelvic-heart fat content, and yield grade. Results from this study suggest that dietary inclusion of a multispecies fungal culture extract might improve the growth performance of beef cattle fed forage-based diets, as well as their carcass characteristics after conclusion of the feedlot finishing phase.

A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei

BackgroundThe well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering.ResultsTo identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter.ConclusionsXTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei.

Physicochemical properties and antimicrobial activities of MytiLec-1, a member from the mytilectin family of mussels

MytiLec-1, the recombinant form of a mussel lectin from Mytillus galloprovincialis, was purified by affinity chromatography and showed the maximum hemagglutination activity at a temperature range of 10 °C to 40 °C and at pH 7.0 to 9.0. Denaturants like urea and acidic-guanidine inhibited its hemagglutination activity significantly. MytiLec-1 was found to be metal-independent though Ca2+ slightly increased the activity of chelated MytiLec-1. The lectin suppressed 65 % growth of Pseudomonas aeruginosa (ATCC 47085) at 200 μg/ml and reduced the formation of biofilm (15 % at 200 μg/ml). Comparing to Shigella sonnei (ATCC 29930), Shigella boydii (ATCC 231903) and Shigella dysenteriae (ATCC 238135), Bacillus cereus (ATCC 14579) was slightly more sensitive to MytiLec-1. At a concentration of 200 μg/disc and 100 μg/ml, MytiLec-1 prevented the growth of Aspergillus niger and agglutinated the spores of Aspergillus niger and Trichoderma reesei, respectively. Amino acid sequences, physicochemical properties and antimicrobial activities of MytiLec-1 were compared with three other lectins (CGL, MTL and MCL from Crenomytilus grayanus, Mytilus trossulas and Mytilus californianus, respectively) from the mytilectin family of bivalve mollusks. It reconfirms the function of these lectins to recognize pathogens and perform important roles in innate immune response of mussels.