Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization.

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5'-CTGGGGTCTGGGG-3' CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

Molecular and bioinformatics analyses reveal two differentially expressed intracellular GH1 ß-glucosidases from the rare alkalophilic fungus Stachybotrys microspora.

The present study reports the isolation and analysis of two novel GH1 ß-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of ß-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (ß/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as ß-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

Portable paper-based colorimetric nanoprobe for the detection of Stachybotrys chartarum using peptide labeled magnetic nanoparticles.

A colorimetric assay is presented for the detection of Stachybotrys chartarum proteases as biomarkers. The assay comprises a gold film acting as solid support and carrying an immobilized peptide substrate that is specific for S. chartarum protease. The substrate was conjugated to black magnetic nanoparticles (MNPs) to form a monolayer on the gold film. Hence, detection nanoprobe is black. If, however, the peptide-MNP fragments are cleaved by S. chartarum proteases present in a sample, the golden color of the detecting nanoprobe becomes apparent so that positive visual readout is enabled. The method was applied to the determination of S. chartarum in (spiked) environmental samples. The limit of detection ranges from 10 to 100 spores·mL-1 depending on the kind of sample (culture, dust, mold and soil). Assay specificity was examined for Aspergillus flavus, Fusarium solani. Penicillin chrysogenum, and Saccharomyces cerevisiae, and negative readouts were observed visually for all samples, except for those also containing S. chartarum. Graphical abstract Schematic presentation of S. chartarum colorimetric nanoprobe.

Recombinant expression of a GH12 ß-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.

The ß-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 ß-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

A novel neutral, halophile Stachybotrys microspora-based endoglucanase active impact on ß-glucan.

The production of cellulases from Stachybotrys microspora strain (A19) has been improved by fed-batch fermentation on Avicel cellulose 10 mg/ml. An endoglucanase EG2 was purified to homogeneity. This cellulase has a molecular mass estimated to 50 kDa when analyzed by a denaturant gel electrophoresis. It exhibited an optimal activity at 50 °C, pH 7.0 and 0.85 M NaCl. Specifically, these results show the thermo-active, alkali-tolerant and halo-tolerant properties of EG2. In addition, this endoglucanase showed its highest activity on barley-ß-glucan, compared to the CMC. Moreover, it was less active on Avicel cellulose. Furthermore, the EG2 activity was stimulated in the presence of EDTA, urea and ß-mercaptoethanol whereas it was reduced in the presence of SDS. This cellulase was highly stable in the presence of organic solvents such as acetone and n-hexane. TLC showed that the main hydrolysis products from EG2 were cellobiose and glucose. This fungal endoglucanase could be potentially important in the conversion of grass-derived biomass into fermentable sugars.

[Expression in Aspergillus niger and characterization of ß-mannanases from Stachybotrys chartarum].

Objective: Using Aspergillus niger as host to express ß-mannanases from Stachybotrys chartarum. Methods: Through sequence analysis of Stachybotrys chartarum genome, two ß-mannanase genes (s16942 and s331) were identified. The primers were designed based on the DNA sequence and the ß-mannanase genes (s16942 and s331) were obtained, and then inserted to the vector pGm. The expression plasmids were transferred into Aspergillus niger. ß-mannanase producing strains (G1-pGm-s16942 and G1-pGm-s331) were isolated after screening several transformants using amdS selection plates and confirmed by PCR fragment sequencing. Results: The molecular weight of the enzymes from G1-pGm-s16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis, and the recombinant proteins did not present in the negative control. Assays of enzymatic property using the crude enzyme preparations indicated that the enzyme from G1-pGm-s16942 exhibited maximum activity (521 U/mL) under the optimum. Conclusion: This was the first study of the heterologous expression of the ß-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the ß-mannanase genes could be expressed successfully with high activities and protein titers.

Investigations on hydrolytic activities from Stachybotrys microspora and their use as an alternative in yeast DNA extraction.

Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, ß-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. ß-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, ß-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.

Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the development and conidiation of fungal pathogens on their hosts and the sensing of host-derived cues. Mycoparasitism is a fungus-fungus interaction comprising host-pathogen cross talk. Until now, only little information is available on the role of the MAPK signaling pathway during this interaction. Here, we report on the differential expression of a MAPK/ERK gene in the mycoparasite Stachybotrys elegans in response to direct parasitism of different vegetative structures of the plant pathogen Rhizoctonia solani (i.e., carbon-rich condition) and to nutrient starvation (i.e., carbon-poor condition). Western blot analysis against ERK1/2 highlighted an increase in their phosphorylated forms when S. elegans was grown under starvation condition compared to that detected in response to mycoparasitism. A higher abundance of phosphorylated ERK1/2 at the third day of interaction compared to that estimated under starvation condition was detected applying LC-MS/MS. At the transcriptional level, smkA, a YERK1 class member, was significantly induced in response to hyphal parasitism compared to parasitized sclerotia at 3, 4, and 5 days of interaction. However, under starvation condition, smkA levels were significantly induced after 7 days of growth. Southern blot analysis revealed that smkA is member of a small gene family. Collectively, these results suggest that smkA could be implicated in the mycoparasitic process in S. elegans as well as in stress-activated pathways. These results may be of wider significance in other fungus-fungus interactions.

Cloning, molecular characterization, and mRNA expression of the thermostable family 3 ß-glucosidase from the rare fungus Stachybotrys microspora.

The filamentous fungus Stachybotrys microspora possess a rich ß-glucosidase system composed of five ß-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the ß-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 ß-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of ß-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/ß)8 triose phosphate isomerase (TIM) barrel, (α/ß)5 sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel ß-glucosidase.

Cellobiose dehydrogenase influences the production of S. microspora ß-glucosidase.

BglG, a Stachybotrys microspora ß-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.

Investigation of the pH-dependent electron transfer mechanism of ascomycetous class II cellobiose dehydrogenases on electrodes.

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on various carbon and thiol-modified gold electrodes. As a result, these systems have been utilized as biocatalyst in biosensors and biofuel cell anodes. Class I CDHs, from basidiomycetous fungi, are highly specific to cellulose or lactose, and DET is only observed at pH values below 5.5. To extend the applicability of CDH-based electrodes, the catalytic properties and the behavior on electrode surfaces of ascomycetous class II CDHs from Chaetomium attrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were investigated. We found that class II CDHs have diverse properties but generally show a lower substrate specificity than class I CDHs by converting also glucose and maltose. Intramolecular electron transfer (IET) and DET at neutral and alkaline pH were observed and elucidated by steady-state kinetics, pre-steady-state kinetics, and electrochemical measurements. The CDHs ability to interact with the electron acceptor cytochrome c and to communicate with electrode surfaces through DET at various pH conditions was used to classify the investigated enzymes. In combination with stopped-flow measurements, a model for the kinetics of the pH-dependent IET is developed. The efficient glucose turnover at neutral/alkaline pH makes some of these new CDHs potential candidates for glucose biosensors and biofuel cell anodes.

Expression of novel ß-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts.

ß-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of ß-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or ß-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for ß-1,3-1,4 glucans, showing maximum activity on barley ß-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley ß-glucan and lichean were 3-O-ß-cellotriosyl-D-glucose and 3-O-ß-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for ß-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.

Hydroxyl distribution in sugar structure and its contributory role in the inhibition of Stachybotrys microspora ß-glucosidase (bglG).

Stachybotrys microspora is a filamentous fungus that produces various ß-glucosidases, of which two have already been characterized. The present study reports on the production of a third one, named bglG, in the presence of d-glucose used as a sole carbon source, and on its subsequent purification and characterization. Although efficiently produced in the presence of d-glucose, bglG continues to be highly inhibited by this sugar. In fact, the addition of d-glucose significantly decreases the glucose formation rates during the hydrolysis of pNPG. This work reports on the effect of various carbohydrates on bglG activity in order to understand the mechanisms adopted by d-glucose to inhibit this enzyme. The findings indicate that bglG is strongly inhibited by d-glucose (44% of the relative activity at 5mM), d-glucitol (96% of the relative activity), d-mannose (56% of the relative activity), cellobiose and maltose (72% and 71% of the relative activity, respectively). On the other hand, d-galactose, d-fructose, lactose, and sucrose have no effect on bglG activity. Similarly, several isomers, such as 2-acetamido-2-deoxy-d-glucose and 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) were noted to bring no change on the relative activity of bglG. d-xylose and xylitol, on the other hand, enhanced bglG activity up to 123% and 120% of relative activity, respectively. Accordingly, the configuration, epimerisation, isomerisation, and substitutions played key roles in bglG inhibition. The effect of the combination of iron (the best activator of bglG, 161%) with some of those additives was also investigated. The findings revealed that, while a combination of iron at a concentration of 10mM with d-glucose resulted in a two-fold decrease in bglG inhibition (84% at 5mM), iron maintained the same effect with the remainder of the additives being tested.

Wheat dehydrin DHN-5 exerts a heat-protective effect on beta-glucosidase and glucose oxidase activities.

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, beta-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 degrees C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.

Mycoparasitism of endophytic fungi isolated from reed on soilborne phytopathogenic fungi and production of cell wall-degrading enzymes in vitro.

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of beta-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of beta-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

A multi-gene phylogeny for Stachybotrys evidences lack of trichodiene synthase (tri5) gene for isolates of one of three intrageneric lineages.

Members of the mitosporic fungal form-genus Stachybotrys, including common indoor contaminants Stachybotrys chartarum, Stachybotrys echinata and Stachybotrys chlorohalonata, are capable of producing potent, protein synthesis-inhibiting, trichothecene mycotoxins. A combined multi-gene approach was used to investigate relationships among species of Stachybotrys against which the presence/absence of the trichothecene biosynthetic pathway gene, trichodiene synthase (tri5), was evaluated. Phylogenetic analyses partitioned species of Stachybotrys into three strongly supported lineages, two of which contained common indoor taxa. No tri5 PCR product was amplified from members of the third clade, which included the only member of the group with a known sexual state, Stachybotrys albipes. Isolates grouped with S. albipes also tested negative for tri5 in Southern analyses. The phylogenetic distribution of tri5 was consistent with known toxin production for the group. For isolates with tri5 product, Bayesian analysis suggested that signal from amino acid determining sites conflicted with the combined phylogeny. Incongruence however, was not supported by either SH-test results or maximum likelihood analyses. Moreover, sites rates analysis showed that tri5 was highly conserved at the amino acid level suggesting that identity at variable sites, among otherwise divergent taxa, might be the result of chance events.

Stachybotrys atra BP-A produces alkali-resistant and thermostable cellulases.

A cellulose-degrading fungal strain has been isolated from a rotten rag. Morphological characterization and ITS1, 5.8S and ITS2 rDNA sequencing showed that the strain is a new isolate of Stachybotrys atra. The strain secreted high cellulase activity in media supplemented with rice straw. However, cellulases were not produced in glucose-supplemented media. The crude cellulase showed the highest activity on amorphous celluloses such as carboxymethyl cellulose, while activity on crystalline celluloses such as Avicel was lower. The optimal temperature and pH for CMCase activity were 70 degrees C and pH 5 respectively, although a second peak of activity was found at pH 8. Activity was strongly inhibited by Cu(2+), Mn(2+) and Hg(2+). Analysis by SDS-PAGE, isoelectric focusing and zymography showed that the strain secretes a complex cellulase system comprising several enzymes. Most of these enzymes are alkali-resistant CMCases that remained stable at pH 9 and 65 degrees C for at least 1 h. Cellulose binding assays showed notable differences among the CMCases. While some CMCase bands did not bind Avicel, other bands bound to this polymer and were eluted either with NaCl or by boiling with SDS. Analysis by two-dimensional electrophoresis showed that the band eluted by SDS boiling contained at least 4 different polypeptides. The complex set of cellulases produced by the strain, and their activity and stability at alkaline pH and a high temperature indicate that both the isolated strain and the cellulases identified are good candidates for biotechnological applications involving cellulose modification.

Purification and biochemical characterization of a transglucosilating beta-glucosidase of Stachybotrys strain.

The filamentous fungus Stachybotrys sp has been shown to possess a rich beta-glucosidase system composed of five beta-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second beta-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly beta-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50 degrees C. The kinetic parameters, K (m) and V (max), on para-nitro-phenyl-beta-D: -glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 +/- 0.11 mM and 211 +/- 0.08 micromol min(-1) ml(-1). One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl beta-glucosidase activity towards salicin, methylumbellypheryl-beta-D: -glucoside (MU-Glc), and p-NPG, it showed a true beta-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.

The role of fungal proteinases in pathophysiology of Stachybotrys chartarum.

The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-alpha than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-beta and TNF-alpha) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 x 10(4) spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.

Use of laccase as a novel, versatile reporter system in filamentous fungi.

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.