Transformation of the medicinal basidiomycete Trametes versicolor to hygromycin B resistance by restriction enzyme mediated integration

Trametes versicolor, a white-rot basidiomycete, degrades cellulose and lignin as well as many recalcitrant chemicals. There have been many reports about the cloning of laccase and peroxidase genes of T. versicolor which are involved in lignin degradation. In order to analyze a gene function and introduce foreign genes into an organism, genetic transformation is required. Here we have successfully transformed T. versicolor to hygromycin B resistance using pAN 7-1 plasmid by restriction enzyme mediated integration and have obtained many mutants in peroxidase activity and growing patterns. The transformation frequency was 25-50 transformants (microg plasmid DNA)(-1). The transformants were quite stable after 10 consecutive transfers in non-selectable medium.     

杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达

以白腐菌杂色云芝Coriolus versicolor RNA为模板,通过RT-PCR获得漆酶Leel基因的cDNA片段。构建了甲醇酵母表达质粒pMETA-Lccl载体,并将其线性化后用电穿孔法导入Pichia methabolica PMAD16,部分阳性克隆的PCR结果表明Lccl基因已经整合到甲醇毕赤酵母染色体上,经摇瓶培养筛选出表达水平较高的酵母工程菌株。漆酶酶活力达53U/L     

Trametes versicolor ligninase: isozyme sequence homology and substrate specificity

The substrate specificity of three ligninase isozymes from the white-rot fungus Trametes versicolor has been investigated using stereochemically defined synthetic dimeric models for lignin. The isozymes have been found to attack non-phenolic beta-O-4 as well as beta-1 lignin model compounds. This finding confirms the classification of the isozymes from T. versicolor as ligninases. The amino-terminal residues of the three isozymes from T. versicolor have been determined using Edman degradation. Minor differences found between the sequences suggest the existence of several structural genes for ligninase in T versicolor. Comparisons have been made with the sequences of three previously reported ligninases from Phanerocompaete chrysosporium, another lignin-degrading fungus. One of the sequences from P. chrysosporium is distinctly more similar to the T. versicolor isozymes than to the other two sequences from P. chrysosporium.     

Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes

Two closely linked lignin peroxidase (LPO)-encoding genes (lpo) from Phanerochaete chrysosporium were isolated. Nucleotide sequence studies indicated that the two genes are separated by 1.3 kb of flanking DNA and transcribed in opposite directions. Cloned P. chrysosporium lpo gene probes have been shown to hybridize to multiple sequences present in the DNAs of the white-rot fungi, Bjerkandera adusta, Coriolus versicolor and Fomes lignosus, but no hybridization was detected with DNA from Pleurotus ostreatus. Thus, lpo gene families appear to be common in a number of lignin-degrading basidiomycetes, some of which have not yet been shown to produce LPO proteins.     

Generation of a transformant showing higher manganese peroxidase (Mnp) activity by overexpression of Mnp gene in Trametes versicolor

Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.     

Bioremediation of paper and pulp mill effluents

Pulp and paper mill effluents pollute water, air and soil, causing a major threat to the environment. Several methods have been attempted by various researchers throughout the world for the removal of colour from pulp and paper mill effluents. The biological colour removal process uses several classes of microorganisms--bacteria, algae and fungi--to degrade the polymeric lignin derived chromophoric material. White rot fungi such as Phanerochaete chrysosporium, Corius versicolor, Trametes versicolor etc., are efficient in decolourizing paper and pulp mill effluents. Gliocladium virens, a saprophytic soil fungus decolourised paper and pulp mill effluents by 42% due to the production of hemicellulase, lignin peroxidase, manganese peroxidase and laccase.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.     

Biodecolorization screening of synthetic dyes by four white-rot fungi in a solid medium: possible role of siderophores

AIMS: Four selected fungi were screened for their ability to decolourize a textile effluent and commercial reactive dyes in a solid medium. METHODS AND RESULTS: Ligninolytic enzymes activities (lignin peroxidase, manganese peroxidase and laccase) and siderophores presence were monitored in decolourized plates. RESULTS: The results showed low lignin peroxidase activity and no manganese peroxidase activity was detected for all fungi. Laccase activity was observed in Reactive Blue 19 decolourized plates by Trametes versicolor and Trametes villosa. Siderophores presence was observed in Trametes versicolor, Phanerochaete chrysosporium and Lentinus edodes decolourized plates. CONCLUSION: Lentinus edodes displayed the greatest decolourization ability both in terms of extent and rapidity of decolourization. SIGNIFICANCE AND IMPACT OF THE STUDY: The transformation observed for dyes open the possibility to study siderophores to treat dyes and textile effluents.     

Cloning and characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor

Six putative lignin peroxidase (LIP) genes were isolated from a lambda EMBL3 phage library of the white-rot fungus, Trametes versicolor, using the Phanerochaete chrysosporium LIP cDNA CLG5 as the probe. Sequence analysis of one of the genes, VLG1, showed that its coding region is interrupted by six small introns (49-64 bp) and that it encodes a mature LIP protein (341 aa; Mr: 36,714) that is preceded by a 25 aa signal sequence. This protein has a relatively high degree of aa homology to the N-termini of the LIP proteins purified from T. versicolor and has an aa homology of 55-60% to the LIP proteins of P. chrysosporium, which is comparable to that found between P. chrysosporium and Phlebia radiata LIP proteins.     

A tomato peroxidase involved in the synthesis of lignin and suberin

The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.     

木质素过氧化物酶基因（LipH8）的克隆及在甲醇毕赤酵母中的表达

以黄孢原毛平革菌 (Phanerochaetechrysosporium)RNA为模板 ,克隆LipH8基因片段 ,研究LipH8基因在甲醇毕赤酵母中的表达。构建了甲醇酵母表达质粒pMETA_LipH8载体 ,并将其线性化后用电穿孔法导入PichiamethabolicaPMAD16 ,部分阳性克隆的PCR结果表明LipH8基因已经整合到甲醇毕赤酵母染色体上 ,经摇瓶培养筛选出表达水平较高的酵母工程菌株。胞外木质素过氧化物酶活力达 932U L。     

Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor

One-electron oxidation activity, as measured by ethylene generation from 2-keto-4-thiomethylbutyric acid, phenol oxidase activity, and the generation of hydroxyl radical were examined in cultures of the lignin-degrading white-rot basidiomycete fungus, Trametes (Coriolus) versicolor. The activity levels of specific lignin-degrading enzymes and cellulases, as well as the rate of wood degradation, also were examined. The fungus secreted a low-molecular-weight substance (M(r) 1000-5000) that catalyzed a redox reaction between molecular oxygen and an electron donor, to produce the hydroxyl radical via hydrogen peroxide. During wood decay, T. versicolor also produced significant amounts of laccase and lignin peroxidase, carboxymethyl cellulase, and Avicelase. The roles of the hydroxyl radical, phenol oxidases, and cellulases in wood degradation by white-rot fungi are discussed. That the hydroxyl radical produced by the low-molecular-weight substance secreted by T. versicolor results in new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase is suggested.     

三种白腐真菌脱色噻嗪染料亚甲基蓝

本研究通过含亚甲基蓝染料的固体培养基,从19株白腐真菌菌株中分离获得3个脱色能力较强的菌株,其在平板上的脱色圈大小分别为7.5cm、6.8cm和5.5cm。鉴定其为：云芝栓孔菌Trametes versicolor（ZT-197）,绒毛栓孔菌Trametes pubescens（ZT-230）和亚黑管孔菌Bjerkandera fumosa（ZT-307）。其中,ZT-230对染料亚甲基蓝的脱色能力最强,可以将染料浓度为50mg/L的100mL液体培养基在6d之内100%脱色,而ZT-197和ZT-307在接种第10天时的脱色率为98%和80%。同时测定了3株白腐真菌在降解染料过程中的漆酶、锰过氧化物酶和木素过氧化物酶3种酶活力的规律：ZT-197和ZT-230均可分泌Lac和MnP两种酶,ZT-307只分泌LiP。本研究说明绒毛栓孔菌ZT-197在印染废水治理方面具有较好的应用前景。     

彩绒革盖菌在猪粪堆肥中应用的初步研究

在以猪粪为原料的静态条垛堆肥的堆体试验中添加了彩绒革盖菌,研究其对堆肥发酵的影响。研究表明彩绒革盖菌在堆肥二次发酵时期有利于堆体温度的提升和保温,说明了在进入堆肥后期彩绒革盖菌对其中剩余的木质素等成分有很好的分解能力,有利于堆肥的腐熟和养分的释放,初步表明彩绒革盖菌是一株理想的堆肥发酵菌株。     

Decoloration of Amaranth by the white-rot fungus Trametes versicolor. Part I. Statistical analysis

The white-rot fungus Trametes versicolor decolorized the mono-azo-substituted naphthalenic dye Amaranth. The relationship between the amount of enzymes present in the system and the efficiency of the decoloration process was investigated. The two responses used to quantify the process of decoloration (i.e., initial decoloration rate, v0, and the percent concentration of dye decolorized in 1 h, %c) were correlated with the amount of three enzymes considered for the study (lignin peroxidase, manganese peroxidase, and laccase) and analyzed through stepwise regression analysis (forward, backward, and mixed). The results of the correlation analysis and those of the regression analysis indicated that lignin peroxidase is the enzyme having the greatest influence on the two responses.     

Decoloration of Amaranth by the white-rot fungus Trametes versicolor. Part II. Verification study

The involvement of lignin peroxidase (LiP) in the decoloration of the mono-azo substituted napthalenic dye Amaranth was investigated with pure enzymes and whole cultures of Trametes versicolor. The verification study confirmed that LiP has a direct influence on the initial decoloration rate and showed that another enzyme, which does not need hydrogen peroxide to function and is not a laccase, also plays a role during decoloration. These results confirm the results of a previous statistical analysis. Furthermore, the fungal mycelium affects the performance of the decoloration process.     

Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus

Biodegradation of endocrine-disrupting bisphenol A was investigated with several white rot fungi (Irpex lacteus, Trametes versicolor, Ganoderma lucidum, Polyporellus brumalis, Pleurotus eryngii, Schizophyllum commune) isolated in Korea and two transformants of T versicolor (strains MrP 1 and MrP 13). I. lacteus degraded 99.4% of 50 mg/l bisphenol A in 3 h incubation and 100% in 12 h incubation. which was the highest degradation rate among the fungal strains tested. T. versicolor degraded 98.2% of 50 mg/l bisphenol A in 12 h incubation. Unexpectedly, the transformant of the Mn-repressed peroxidase gene of T. versicolor, strain MrP 1, degraded 76.5% of 50 mg/l bisphenol A in 12 h incubation, which was a lower degradation rate than wild-type T. versicolor. The removal of bisphenol A by I. lacteus occurred mainly by biodegradation rather than adsorption. Optimum carbon sources for biodegradation of bisphenol A by I. lacteus were glucose and starch, and optimum nitrogen sources were yeast extract and tryptone in a minimal salts medium; however, bisphenol A degradation was higher in nutrient-rich YMG medium than that in a minimal salts medium. The initial degradation of endocrine disruptors was accompanied by the activities of manganese peroxidase and laccase in the culture