| Phanerochaete chrysosporium is a lignin degrading white rot basidiomycete fungus. There is considerable interest in this organism due to its applications in biopulping, xenobiotic degradation and in bioconversion of lignocellulosic materials to feeds, fuels, and chemicals. Hence, the physiology, genetics, and molecular biology of this organism is being studied extensively. To further the molecular genetic analysis of this fungus, we constructed vector pRR12 that transforms P. chrysosporium to G418 resistance. Vector pG12-1, a derivative pRR12, was isolated from a pRR12 transformant and was also shown to transform P. chryosoporium. This vector appeared to result from a rearrangement between pRR12 and an endogenous plasmid(pME). Vector pG12-1 was shown to be maintained in P. chrysosporium in a low copy, circular, extrachromosomal state and was recovered intact from fungal transformants by E. coli transformation. The vector was stably transmitted to basidiospore progeny of the transformants. A similar mode of transformation was found to occur with two other vectors, YIp5-kan{dollar}sp{lcub}rm r{rcub}{dollar} and pAN7-1, which transform P. chrysosporium to G418 and hygromycin resistance, respectively. Plasmid p12-6, a common derivative of pG12-1 and YIp5-kan{dollar}sp{lcub}rm r{rcub}{dollar}, was also shown to transform P. chrysosporium. Southern blot analysis of p12-6 transformants shows directly that the vector is maintained extrachromosomally, although not autonomously. Two classes of plasmids (p12-6 and p511), were found to be recovered by E. coli transformation using total DNA of p12-6 transformants. The lacZ gene inserted in to p12-6, was shown to be passed intact through P. chrysosporium suggesting that p12-6 has a potential as a shuttle vector. When the kan{dollar}sp{lcub}rm r{rcub}{dollar} determinant was inserted into the coding region of a genomic clone containing the lignin peroxidase H2, the resulting plasmid was shown to transform P. chrysosporium to G418 resistance, was maintained in a high copy number autonomous form in the transformants, and was recoverable by E. coli transformation. |
