A complex metabolic network and its biomarkers regulate laccase production in white-rot fungus Cerrena unicolor 87613.

BACKGROUND: White-rot fungi are known to naturally produce high quantities of laccase, which exhibit commendable stability and catalytic efficiency. However, their laccase production does not meet the demands for industrial-scale applications. To address this limitation, it is crucial to optimize the conditions for laccase production. However, the regulatory mechanisms underlying different conditions remain unclear. This knowledge gap hinders the cost-effective application of laccases. RESULTS: In this study, we utilized transcriptomic and metabolomic data to investigate a promising laccase producer, Cerrena unicolor 87613, cultivated with fructose as the carbon source. Our comprehensive analysis of differentially expressed genes (DEGs) and differentially abundant metabolites (DAMs) aimed to identify changes in cellular processes that could affect laccase production. As a result, we discovered a complex metabolic network primarily involving carbon metabolism and amino acid metabolism, which exhibited contrasting changes between transcription and metabolic patterns. Within this network, we identified five biomarkers, including succinate, serine, methionine, glutamate and reduced glutathione, that played crucial roles in co-determining laccase production levels. CONCLUSIONS: Our study proposed a complex metabolic network and identified key biomarkers that determine the production level of laccase in the commercially promising Cerrena unicolor 87613. These findings not only shed light on the regulatory mechanisms of carbon sources in laccase production, but also provide a theoretical foundation for enhancing laccase production through strategic reprogramming of metabolic pathways, especially related to the citrate cycle and specific amino acid metabolism.

Efficient crop straws biotreatment using the fungus Cerrena Unicolor GC.u01.

Lignin is main composition of agricultural biomass which can be decomposed through enzymatic hydrolysis by fungi. However, there are still needs to identify more efficient and effective fungal stain for biomass valorization. In this study, lignin degrading fungi from birch forest were screened for sustainable degradation of waste agricultural straws. The most effective strain was identified as Cerrena unicolor GC.u01 using 18 S rDNA gene-sequencing technology. Three different crop straws (corn stalk, rice and wheat straws) were used for the biotreatment studies. The activities of lignin degrading enzymes, laccase (Lac), cellulase and xylanase, secreted by C. unicolor were also determined. Scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and thermal gravimetric analyzer (TGA) were further used to monitor the effects of the biotreatment process. The results showed that C. unicolor degraded 34.3% rice straw lignin, a percentage which was higher than other isolated strains after 15 d straw liquid fermentation. The highest Lac activity (8.396 U•mL- 1) was observed with corn stalk on the 7 d. Cellulase and xylanase activities, in the same biomass, were higher than those of wheat and rice straws after 15 d. Furthermore, SEM, FTIR and TGA analyses showed that C. unicolor pretreatment process had significant effects on corn stalk, rice and wheat straws' structures. The newly isolated stain of C. unicolor demonstrated high lignin degradation potential that can provide effective, ecofriendly means of valorizing biomass to industrial useable raw-material.

Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Genome-wide study of Cerrena unicolor 87613 laccase gene family and their mode prediction in association with substrate oxidation.

BACKGROUND: Laccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1). RESULTS: In this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates. CONCLUSIONS: Our findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor.

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Convenient Tool to Assess the Potential of Low Molecular Fraction Derived from Medicinal Fungus Cerrena unicolor in Action on L929 and CT-26 Cell Lines.

The increase in the incidence of cancer has contributed to the search for new therapeutic methods. In recent years, the use of preparations of natural origin from medical fungi has increased. One such active substance is the extracellular, low molecular active fraction obtained from the medicinal fungus Cerrena unicolor. This study aimed to monitor the pharmacokinetics of different concentrations of substances isolated from the medicinal fungus Cerrena unicolor (ex-LMS) using the ECIS technique. In the study, mouse L929 fibroblasts and colon cancer CT26 cell lines were treated with different concentrations of the active fractions obtained from Cerrena unicolor: C1 = 2.285 (µg/mL); C2 = 22.85 (µg/mL); and C3 = 228.5 (µg/mL). This study demonstrated that the tested preparation from Cerrena unicolor had no considerable effect on the resistance, capacitance, and impedance of L929 fibroblast cells, which was an indicator of no significant effect on its physiological processes. At the same time, those parameters exhibited a decrease in colon cancer cell viability. Following our previous and current studies on Cerrena unicolor, ex-LMS extracts can be safely used in anticancer therapy or chemoprevention with no significant harmful effects on normal cells.

Genomic Studies of White-Rot Fungus Cerrena unicolor SP02 Provide Insights into Food Safety Value-Added Utilization of Non-Food Lignocellulosic Biomass.

Cerrena unicolor is an ecologically and biotechnologically important wood-degrading basidiomycete with high lignocellulose degrading ability. Biological and genetic investigations are limited in the Cerrena genus and, thus, hinder genetic modification and commercial use. The aim of the present study was to provide a global understanding through genomic and experimental research about lignocellulosic biomass utilization by Cerrena unicolor. In this study, we reported the genome sequence of C. unicolor SP02 by using the Illumina and PacBio 20 platforms to obtain trustworthy assembly and annotation. This is the combinational 2nd and 3rd genome sequencing and assembly of C. unicolor species. The generated genome was 42.79 Mb in size with an N50 contig size of 2.48 Mb, a G + C content of 47.43%, and encoding of 12,277 predicted genes. The genes encoding various lignocellulolytic enzymes including laccase, lignin peroxidase, manganese peroxidase, cytochromes P450, cellulase, xylanase, α-amylase, and pectinase involved in the degradation of lignin, cellulose, xylan, starch, pectin, and chitin that showed the C. unicolor SP02 potentially have a wide range of applications in lignocellulosic biomass conversion. Genome-scale metabolic analysis opened up a valuable resource for a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases that provide insights into the genetic basis and molecular mechanisms for lignocellulosic degradation. The C. unicolor SP02 model can be used for the development of efficient microbial cell factories in lignocellulosic industries. The understanding of the genetic material of C. unicolor SP02 coding for the lignocellulolytic enzymes will significantly benefit us in genetic manipulation, site-directed mutagenesis, and industrial biotechnology.

Thromboelastometric Analysis of Anticancer Cerrena unicolor Subfractions Reveal Their Potential as Fibrin Glue Drug Carrier Enhancers.

In this study, the influence of two subfractions (with previously proven anti-cancer properties) isolated from wood rot fungus Cerrena unicolor on the formation of a fibrin clot was investigated in the context of potential use as fibrin glue and sealant enhancers and potential wound healing agents. With the use of ROTEM thromboelastometry, we demonstrated that, in the presence of fibrinogen and thrombin, the S6 fraction accelerated the formation of a fibrin clot, had a positive effect on its elasticity modulus, and enhanced the degree of fibrin cross-linking. The S5 fraction alone showed no influence on the fibrin coagulation process; however, in the presence of fibrin, it exhibited a decrease in anti-proliferative properties against the HT-29 line, while it increased the proliferation of cells in general at a concentration of 100 µg/mL. Both fractions retained their proapoptotic properties to a lesser degree. In combination with the S6 fraction in the ratio of 1:1 and 1:3, the fractions contributed to increased inhibition of the activity of matrix metalloproteinases (MMPs). This may suggest anti-metastatic activity of the combined fractions. In conclusion, the potential of the fractions isolated from the C. unicolor secretome to be used as a means of improving the wound healing process was presented. The potential for delivering agents with cytostatic properties introduced far from the site of action or exerting a pro-proliferative effect at the wound site with the aid of a fibrin sealant was demonstrated.

Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase's transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Antimicrobial Activity and Barrier Properties against UV Radiation of Alkaline and Enzymatically Treated Linen Woven Fabrics Coated with Inorganic Hybrid Material.

One of the directions of development in the textiles industry is the search for new technologies for producing modern multifunctional products. New solutions are sought to obtain materials that will protect humans against the harmful effects of the environment, including such factors as the activity of microorganisms and UV radiation. Products made of natural cellulose fibers are often used. In the case of this type of material, it is very important to perform appropriate pretreatment before subsequent technological processes. This treatment has the aim of removing impurities from the surface of the fibers, which results in the improvement of sorption properties and adhesion, leading directly to the better penetration of dyes and chemical modifiers into the structure of the materials. In this work, linen fabrics were subjected to a new, innovative treatment being a combination of bio-pretreatment using laccase from Cerrena unicolor and modification with CuO-SiO2 hybrid oxide microparticles by a dip-coating method. To compare the effect of alkaline or enzymatic pretreatment on the microstructure of the linen woven fabrics, SEM analysis was performed. The new textile products obtained after this combined process exhibit very good antimicrobial activity against Candida albicans, significant antibacterial activity against the Gram-negative Escherichia coli and the Gram-positive Staphylococcus aureus, as well as very good UV protection properties (ultraviolet protection factor (UPF) > 40). These innovative materials can be used especially for clothing or outdoor textiles for which resistance to microorganisms is required, as well as to protect people who are exposed to long-term, harmful effects of UV radiation.

A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization.

Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.

Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor.

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2' -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

Combined Effect of Light and Nutrients on the Micromorphology of the White rot Fungus Cerrena Unicolor.

Light influences developmental pathways in fungi. Recent transcriptomic and biochemical analyses have demonstrated that light influences the metabolism of a white-rot basidiomycete Cerrena unicolor. However, the expression profile of genes involved in the growth and development, or micromorphological observations of the mycelium in response to variable lighting and culturing media, have not performed. We aim to reveal the effect of light and nutrients on C. unicolor growth and a potential relationship between the culture medium and lighting conditions on fungus micromorphological structures. Confocal laser scanning microscopy and scanning electron microscopy were employed for morphological observations of C. unicolor mycelium cultivated in red, blue, green, and white light and darkness on mineral and sawdust media. A comprehensive analysis of C. unicolor differentially expressed genes (DEGs) was employed to find global changes in the expression profiles of genes putatively involved in light-dependent morphogenesis. Both light and nutrients influenced C. unicolor growth and development. Considerable differences in the micromorphology of the mycelia were found, which were partially reflected in the functional groups of DEGs observed in the fungus transcriptomes. A complex cross-interaction of nutritional and environmental signals on C. unicolor growth and morphology was suggested. The results are a promising starting point for further investigations of fungus photobiology.

Comparison of downstream processing methods in purification of highly active laccase.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

RNA Sequencing Reveals Differential Gene Expression of Cerrena Unicolor in Response to Variable Lighting Conditions.

To elucidate the light-dependent gene expression in Cerrena unicolor FCL139, the transcriptomes of the fungus growing in white, blue, green, and red lighting conditions and darkness were analysed. Among 10,413 all-unigenes detected in C. unicolor, 7762 were found to be expressed in all tested conditions. Transcripts encoding putative fungal photoreceptors in the C. unicolor transcriptome were identified. The number of transcripts uniquely produced by fungus ranged from 20 during its growth in darkness to 112 in the green lighting conditions. We identified numerous genes whose expression differed substantially between the darkness (control) and each of the light variants tested, with the greatest number of differentially expressed genes (DEGs) (454 up- and 457 down-regulated) observed for the white lighting conditions. The DEGs comprised those involved in primary carbohydrate metabolism, amino acid metabolism, autophagy, nucleotide repair systems, signalling pathways, and carotenoid metabolism as defined using Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The analysis of the expression profile of genes coding for lignocellulose-degrading enzymes suggests that the wood-degradation properties of C. unicolor may be independent of the lighting conditions and may result from the overall stimulation of fungal metabolism by daylight.

Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly.

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

Comparative transcriptomic analysis of Cerrena unicolor revealed differential expression of genes engaged in degradation of various kinds of wood.

To explore the number of enzymes engaged by Cerrena unicolor FCL139 for wood degradation, the transcriptomes of the fungus growing on birch, ash, maple sawdust and the control liquid medium were analyzed. Among 12,966 gene models predicted for the C. unicolor genome, 10,396 all-unigenes were detected, of which 9567 were found to be expressed in each of the tested growth media. The highest number (107) of unique transcripts was detected during fungus growth in the control liquid medium, while the lowest number (11) - in the fungal culture comprising maple saw dust. Analysis of C. unicolor transcriptomes identified numerous genes whose expression differed substantially between the mycelia growing in control medium and each of the sawdust media used, with the highest number (828) of upregulated transcripts observed during the fungus growth on the ash medium. Among the 294 genes that were potentially engaged in wood degradation, the expression of 59 was significantly (p < .01) changed in the tested conditions. The transcripts of 37 of those genes were at least four times more abundant in the cells grown in all sawdust media when compared to the control medium. Upregulated genes coding for cellulases and, to a lower extent, hemicellulases predominated during fungus growth on sawdust. Transcripts encoding cellulolytic enzymes were the most abundant in mycelia grown on birch and maple while lower number of such transcripts was detected in fungus growing on ash. The expression pattern of lignolytic activities-coding genes was strongly dependent on the type of sawdust applied for fungus growth medium.

Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01.

A novel laccase was purified from fermentation broth of the white rot fungus Cerrena unicolor strain GSM-01 following three ion-exchange chromatography steps and one gel-filtration step. The purified enzyme was determined to be a monomeric protein of 63.2kDa and demonstrated high oxidation activity of 2.05×104U/mg towards ABTS. Its cDNA, gene, and amino acid sequences were obtained. It possessed high sequence similarity with that of other laccases but different enzymatic properties. It manifested optimal pH and temperature of 2.6 and 45°C, respectively. Fe3+ and Fe2+ were the most efficient inhibitors towards Cerrena unicolor laccase (CUL), while Mn2+ can slightly enhance the laccase activity of 3.8-10.5%. The Km and Vmax of CUL were estimated to 302.7µM and 13.6µMm-1, respectively. CUL was effective in the decolorization of bromothymol blue, evans blue, methyl orange, and malachite green with decolorization efficiencies of 50%-85%. It possesses potential application in textile and environmental industries.

Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells.

Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.