Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Biotechnology for Biofuels

Timo Schuerg¹  Steven W. Singer¹  Jennifer Gorman¹  Pallas Chou²  Marina Jecmenica³  Carlos Romero-Vazquez⁴  Julia Prinz⁵  Lena Floerl⁵  Susanne Fritsche⁶  Simon Harth⁷  Raphael Gabriel⁸  Linda Matz⁸  Anne Oostlander⁸  Laure Curran⁹  André Fleißner^1,10 

[1] Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;American High School, 36300 Fremont Blvd, 94536, Fremont, CA, USA;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190, Vienna, Austria;Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;College of Natural Sciences, University of Puerto-Rico, Rio Pedras, 17 Ave. Universidad STE 1701, 00925, San Juan, Puerto Rico, USA;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Department of Food Science and Technology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Frankfurt Institute of Molecular Biosciences, Goethe University Frankfurt, 60438, Frankfurt Am Main, Germany;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;Institut für Genetik, Technische Universität Braunschweig, Brunswick, Germany;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 94720, Berkeley, CA, USA;Joint BioEnergy Institute, 5885 Hollis Street, 94608, Emeryville, California, United States;École Polytechnique Fédérale de Lausanne, 1015, Lausanne, Vaud, Switzerland;Institut für Genetik, Technische Universität Braunschweig, Brunswick, Germany;

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

DOI  :  10.1186/s13068-020-01804-x

来源: Springer

【 摘 要 】

BackgroundFungal 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.ResultsHere, 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.ConclusionThe 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】

CC BY