Genetic transformation and fluorescent protein expression in the biomaterial-producing fungus Fomes fomentarius

Background Fungal-based materials are currently studied as renewable and sustainable alternatives to fossil-based materials used in construction, packaging, textile, and fashion industries. The polypore fungus Fomes fomentarius produces a range of lightweight, durable, and flexible materials from diverse plant residual streams. Genetic engineering of this fungus is an important technological objective, both from fundamental perspectives and to optimize performance characteristics of F. fomentarius materials. Results We provide proof of principle that F. fomentarius can be genetically transformed. Hyphae produced high amounts of protoplasts using standard protocols. These protoplasts were transformed using nourseothricin selection and a nourseothricin N-acetyl transferase resistance marker ( natR ). Expression of natR was successful using a F. fomentarius translation elongation factor promoter. Stable, multicopy, and non-targeted integration of exogenous cassettes with recipient genome was confirmed by diagnostic PCR and Southern blotting. Preliminary attempts at genome editing based disruption of a putative ku70 encoding gene were, however, unsuccessful. We validated expression of two fluorescent proteins using a Schizophyllum commune H2B promoter using qRT-PCR and fluorescent microscopy. These data confirm functionality of mCherry and eGFP in F. fomentarius and suggest these proteins can be used for future protein localisation studies. Finally, we report DNase activity in F. fomentarius which we hypothesize may be a significant technical challenge for genetic transformation of this fungus. Conclusion F. fomentarius can be genetically transformed using the promoters, marker, and nourseothricin selection system developed in this study. Expression of both eGFP and mCherry is a significant step towards cell biological and gene functional analyses using fluorescent microscopy. We have laid the foundations for overexpression studies, and, additionally, identified several challenges which need to be overcome for targeted genetic engineering in this important material producing fungus.