Establishment of an antimetabolite-based transformation system for the wood-decaying basidiomycete <i>Phanerochaete chrysosporium</i>

The model wood-decaying basidiomycete <i>Phanerochaete chrysosporium</i> 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 <i>P. chrysosporium</i>. The transformation system utilizes pyrithiamine (PT), a thiamine antimetabolite, in combination with the pPTRII vector that contains the PT resistance gene (<i>ptrA</i>). PT effectively inhibited the growth of <i>P. chrysosporium</i>, and the introduction of <i>ptrA</i> 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 <i>actin1</i>, <i>DED</i>, and <i>GAPDH</i> genes. Promoter activities were assessed via fluorescent microscopy observation of transformant mycelia and GFP quantification in crude cell extracts, revealing that the <i>actin1</i> 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 <i>P. chrysosporium</i> 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 <i>P. chrysosporium</i>.IMPORTANCEWood-decaying basidiomycetes are well-recognized for their exceptional capabilities to decompose lignocellulosic biomass and oxidize a broad range of complex organic compounds. These capabilities are essential for maintaining the forest ecosystem and hold potential in biotechnological applications such as transforming recalcitrant biomass into useful compounds and degrading toxic substances in industrial effluents. However, genetic manipulation in basidiomycetes remains challenging because of the inefficiency of transformation systems. In the model lignocellulose-degrading basidiomycete, <i>P. chrysosporium,</i> transformation methods using dominant markers are scarce and were reported over two decades ago, necessitating the re-establishment of a functional system compatible with modern genetic tools like genome editing technology. In this study, an efficient genetic transformation system was achieved by using an antimetabolite-based selection strategy for <i>P. chrysosporium</i>. This transformation system would lay the foundation for advancing our understanding of the molecular mechanisms of wood decomposition and support the targeted optimization of basidiomycetes for various biotechnological applications.