Gabriel Raphael, Prinz Julia, Jecmenica Marina, Romero-Vazquez Carlos, Chou Pallas, Harth Simon, Floerl Lena, Curran Laure, Oostlander Anne, Matz Linda, Fritsche Susanne, Gorman Jennifer, Schuerg Timo, Fleißner André, Singer Steven W
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 USA.
Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, California 94608 United States.
Biotechnol Biofuels. 2020 Oct 10;13:167. doi: 10.1186/s13068-020-01804-x. eCollection 2020.
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 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.
Here, we report -mediated transformation (ATMT) of using the 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 , 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 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.
The genetic tools developed for can now be used individually or in combination to further improve thermostable enzyme production by this fungus.
真菌酶对工业生物技术至关重要，包括将植物生物质转化为生物燃料和生物基化学品。近年来，人们对使用嗜热真菌的酶越来越感兴趣，与目前使用的真菌酶相比，嗜热真菌的酶通常具有更高的反应速率和耐热性。嗜热丝状真菌能产生大量高度耐热的植物细胞壁降解酶。然而，尚未为这种真菌开发出遗传工具，这阻碍了菌株工程的研究。本研究的目的是开发诸如转化系统、CRISPR/Cas9基因编辑系统和有性杂交方案等菌株工程工具，以提高酶的产量。
在此，我们报道了使用潮霉素B抗性标记基因对[具体真菌名称未给出]进行农杆菌介导的转化（ATMT）。对新开发的转化方案进行了优化，并用于整合转录木聚糖酶调节因子[具体名称未给出]的表达盒，这导致木聚糖酶活性提高了500%。此外，在这种真菌中建立了CRISPR/Cas9基因编辑系统，并测试了两种不同的引导RNA（gRNA）分别以10%和35%的缺失效率删除[具体基因名称未给出]的同源物。最后，使用潮霉素B抗性和5-氟乳清酸抗性亲本菌株建立了有性杂交方案。在一周内完成了在选择性培养基上的杂交和子代分离。
为[具体真菌名称未给出]开发的遗传工具现在可以单独使用或组合使用，以进一步提高这种真菌的耐热酶产量。
