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

Abstract

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 (Wg plasmid DNA)31 . The transformants were quite stable after 10 consecutive transfers in non-selectable medium. ß

Genetic introduction of foreign genes to Pleurotus eryngii by restriction enzyme-mediated integration

“…The tiny colonies around the big colonies turned out to be satellite colonies that could not grow on new hygromycin plates, thereby indicating that they had originated from untransformed host protoplasts. The transformation yield was 10-40 transformants per µg of plasmid DNA, which was coincident with previous reports concerning mushroom transformation (Kim et al, 2002(Kim et al, , 2004Joh et al, 2003). In order to verify whether the transformants harbored the plasmid-originated DNA, the genomic DNAs of the 5 selected transformants and the original strain as a control were extracted and subjected to PCR analysis targeting the ECFP gene using the primer set utilized for the construction of pAN7-1-ECFP.…”

“…Meanwhile, REMI has also proven a powerful tool for the transformation of a variety of microorganisms, including Ustilago maydis (Bölker et al, 1995), Dictyostelium (Kuspa and Loomis, 1992), and Trichoderma (Wang et al, 2009). This method has been shown to be particularly efficient for the transformation of a variety of mushrooms, including Pleurotus ostreatus (Irie et al, 2001;Joh et al, 2003), Trametes versicolor (Kim et al, 2002), Ganoderma lucidum (Kim et al, 2004), and Schizophyllum commune (van Peer et al, 2009). However, an efficient method for the introduction of foreign genes to the P. eryngii genome has yet to be developed.…”

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

“…These results also explain the unsuccessful attempts at Agrobacterium-mediated transformations of C. passeckerianus with pGR4iGM3, pGR4-GFP, pGreenhph004, and pBIN7-1, all of which contain this same truncated hph gene. In contrast, truncated hph-containing plasmids were able to generate the hygromycin-resistant transformants of A. bisporus, H. cylindrosporum, P. ostreatus, S. commune, and T. versicolor (29,34,35,43,55), pointing out the variations among different basidiomycete species.…”

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

“…Trametes versicolor is a white-rot basidiomycete that has high degrading ability against a wide variety of materials, and we have isolated a T. versicolor strain that has degrading abilities towards many recalcitrant materials including aromatic hydrocarbons (Song, 1997), explosives (Cheong et al, 2006) and phenanthrene (Han et al, 2004). Therefore, we evaluated this strain of T. versicolor using a genetic transformation system (Kim et al, 2002), and cDNA of manganese-repressed peroxidase (Kim et al, 2005) and manganese-dependent peroxidase (MnP; Yeo et al, 2007) were cloned and evaluated. A laccase activity and its transcript level were increased during the degradation of TNT and its catabolic intermediates by T. versicolor (Cheong et al, 2006).…”