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

A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading filamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 ori, 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 III and dam methylation analyses, respectively, indicated that pG12-I 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.