Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Biotechnology for Biofuels

Timo Schuerg¹  Pallas Chou¹  Laure Curran¹  Carlos Romero-Vazquez¹  Marina Jecmenica¹  Jennifer Gorman¹  Julia Prinz¹  Lena Floerl¹  Anne Oostlander¹  Raphael Gabriel¹  Linda Matz¹  Susanne Fritsche¹  Simon Harth¹  Steven W. Singer¹  André Fleißner² 

[1] Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory;Institut für Genetik, Technische Universität Braunschweig;

关键词: Filamentous fungi;    Thermoascus aurantiacus;    Agrobacterium tumefaciens;    Genetic transformation;    CRISPR/Cas9 system;    Sexual crossing;   

DOI  :  10.1186/s13068-020-01804-x

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown