Development of a Transposon-Based Genome Engineering Toolkit for Efficient and Adaptable Genetic Modifications in <i>Wolfiporia cocos</i>

Advances in genome engineering of fungal strains are rapidly progressing, driven by the increasing interest in fungal biotechnology. Given the unique genomic and cellular complexity of fungi, each strain benefits from a tailored toolkit for efficient genome engineering. Here, we present a transposon-based engineering toolkit specifically optimized for <i>Wolfiporia cocos</i>, a species valued for its bioactive compounds. This toolkit significantly improves transformation efficiency, enabling multiplexed gene integration and facilitating rapid, flexible prototyping by assembling multiple genes into transposomes in a cocktail format, which bypasses the need for an intricate genetic circuit assembly. Engineered strains demonstrated stable expression across generations, as confirmed by successful genomic integration. Additionally, we identified six native <i>W. cocos</i> promoters from transcriptomic data, with two showing robust, constitutive expression in the mycelium of engineered strains. This transposon-based toolkit offers a versatile resource for synthetic biology, supporting efficient and adaptable genetic modifications within fungal systems.