A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium

Abstract

A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading ifiamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 on, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P. chrysosporium. Vector pG12-1 was able to transform P. chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation. Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Exonuclease IH and dam methylation analyses, respectively, indicated that pG12-1 undergoes replication in P. chrysosporium and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long-term nonselective growth. There is no evidence for integration of the vector into the chromosome at any stage.In recent years, research has intensified worldwide on the ligninolytic fungus Phanerochaete chrysosporium because of the industrial potential of this organism in biopulping, in the conversion of lignin and lignocellulosic materials to feeds, fuels, and chemicals (22, 47), and in detoxifying recalcitrant environmental pollutants such as dioxins, polychlorinated biphenyls, and benzo(a)pyrenes (10, 11). Lignin peroxidases and manganese peroxidases, two families of extracellular, glycosylated, heme proteins involved in lignin degradation by P. chrysosporium, have been isolated and characterized (16, 22,47,48). Genetic studies with this organism have been limited, but auxotrophic mutants (15,26,27) and mutants lacking various secondary metabolic activities (7, 21, 24) have been isolated. Both cDNA (13,49,53,55) and genomic clones (3,5,8,20,41,42,52,54) for lignin peroxidases and cDNA clones for manganese peroxidases (31, 34) have been isolated and sequenced. We previously described vector pRR12, which was shown to transform P. chrysosporium to G418 resistance and was rescued in Escherichia coli from the total DNA of pRR12 transformants (35). Alic et al. (1, 2) have described an integrative transformation system in which a plasmid vector carrying either an heterologous ADE2 or ADE5 gene from Schizophyllum commune was used to complement the appropriate adenine auxotroph of P. chrysosporium. We describe here a novel transformation vector for P. chrysosporium that is maintained stably in the transformants in an extrachromosomal circular form and is readily recoverable from the total DNA of transformants. MATERIALS AND METHODSStrains and media. P. chrysosporium ME446 (ATCC 34541) was maintained as previously described (21). E. coli DHSa [F-endAl hsdR17(rK-MK+) supE44 thi-J deoR recAl gyrA96 relAIA (argF-lacZYA)U169 4i80dlacZAMJSA-], was used for the maintenance of plasmid vectors. Gold...

Genetic damage following introduction of DNA in Phycomyces

“…Rearranged plasmids have been found repeatedly in the Mucorales (Burmester, 1992(Burmester, , 1995Burmester et al, 1990;Schilde et al, 2001;Yanai et al, 1991), as in other filamentous fungi (Bej and Perlin, 1991;Randall et al, 1991). The most common changes consist in losses and repetitions of plasmids sequences, but there have been cases of acquisitions.…”

Autonomous Plasmid Replication inAspergillus nidulans:AMA1 and MATE Elements

“…Although plasmid replicators have been isolated from various fungi (Garcia-Pedrajas and Roncero, 1996;Tsukuda et al, 1988;Randall et al, 1991;Peng et al, 1993;and others), most of them have been found accidentally when cloning unrelated genes. The scarcity of such sequences, as well as other considerations, suggests that chromosomal replicators alone are insufficient for plasmid replication.…”

Enzymology and Molecular Biology of Lignin Degradation

“…For P. chrysosporium, methodology has been established for auxotroph production (Gold et al 1982), recombination analysis (Alic and Gold 1985;Raeder et al 1989b;Krejci and Homolka 1991;Gaskell et al 1994), rapid DNA and RNA purification (Haylock et al 1985;Raeder and Broda 1985), differential display (Birch 1998;Kurihara et al 2002;Assmann et al 2003), pulsed field electrophoretic karyotyping (Gaskell et al 1991;D'Souza et al 1993;Orth et al 1994), and genetic transformation by auxotroph complementation (Alic et al 1989(Alic et al ,1990(Alic et al ,1991Alic 1990;Randall et al 1991;Akileswaran et al 1993;Zapanta et al 1998) and by drug resistance markers (Randall et al 1989(Randall et al ,1991Randall and Reddy 1992;Gessner and Raeder 1994;Ma et al 2003). Transformation efficiencies are relatively low and gene disruptions are difficult (Alic et al 1993), but reporters for studying gene expression have been described (Gettemy et al 1997;Birch et al 1998;Ma et al 2001).…”