Establishment of an antimetabolite-based transformation system for the wood-decaying basidiomycete Phanerochaete chrysosporium

The model wood-decaying basidiomycete Phanerochaete chrysosporium has been extensively studied to elucidate the molecular mechanisms of wood decomposition. However, genetic studies have been limited by the lack of adequate genetic tools. Here, we established an antimetabolite-based transformation system, originally developed for ascomycetes, for use in P. chrysosporium. The transformation system utilizes pyrithiamine (PT), a thiamine antimetabolite, in combination with the pPTRII vector that contains the PT resistance gene (ptrA). PT effectively inhibited the growth of P. chrysosporium, and the introduction of ptrA conferred resistance to transformant mycelia. The transformation efficiency was comparable to that in ascomycetes, suggesting that the transformation system is also applicable to basidiomycetes. To examine the suitability of the system for heterologous gene expression, four cassettes were constructed to express GFP under the promoters of the actin1, DED, and GAPDH genes. Promoter activities were assessed via fluorescent microscopy observation of transformant mycelia and GFP quantification in crude cell extracts, revealing that the actin1 promoter drove the highest level of expression. Furthermore, truncating repeat sequences of the autonomously replicating sequence in the vector backbone improved transformation efficiency, likely due to the reduction in vector size. The transformation efficiency of the gene cassette-inserted vector in P. chrysosporium was relatively higher than that reported with alternative transformation systems in other species of wood- decaying basidiomycetes. The present transformation system could provide a platform for protein expression and genetic engineering in P. chrysosporium and potentially in other wood-decaying basidiomycetes.