Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus
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Biotechnology for Biofuels Open Access
RESEARCH
Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus Raphael Gabriel1,2,3*, Julia Prinz1,2,4, Marina Jecmenica1,2,5,6, Carlos Romero‑Vazquez1,2,7, Pallas Chou1,2,8, Simon Harth1,2,9, Lena Floerl1,2,4, Laure Curran1,2,10, Anne Oostlander1,2,3, Linda Matz1,2,3, Susanne Fritsche1,2,11, Jennifer Gorman1,2, Timo Schuerg1,2, André Fleißner3 and Steven W. Singer1,2*
Abstract Background: Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results: Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion: The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus. Keywords: Filamentous fungi, Thermoascus aurantiacus, Agrobacterium tumefaciens, Genetic transformation, CRISPR/ Cas9 system, Sexual crossing, Xylanases, Enzyme production Background Due to the potentially deleterious impacts of climate change, which is mainly caused by the use of fossil resources, great efforts have been made to explore the applicability of lignocellulosic plant biomass as *Correspondence: [email protected]; [email protected] 1 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA Full list of author information is available at the end of the article
sustainable alternative to fossil fuels. Lignocellulosic biomass is the most abundant organic material on earth, consisting primarily of the sugar polymers cellulose and hemicellulose and the aromatic polymer lignin [1, 2
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