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. 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. The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

中文翻译：

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嗜热丝状真菌 Thermoascus aurantiacus 遗传工具的开发

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，人们越来越关注使用来自嗜热真菌的酶，与目前使用的真菌酶相比，它们通常具有更高的反应速率和耐热性。嗜热丝状真菌 Thermoascus aurantiacus 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遗传工具，这阻碍了菌株工程的努力。本研究的目标是开发菌株工程工具，例如转化系统、CRISPR/Cas9 基因编辑系统和有性杂交协议，以提高酶的产量。在这里，我们报告了根癌农杆菌介导的 T. aurantiacus 使用 hph 标记基因，赋予对潮霉素 B 的抗性。新开发的转化方案经过优化并用于整合转录木聚糖酶调节剂 xlnR 的表达盒，这导致木聚糖酶活性增加高达 500%。此外，在该真菌中建立了 CRISPR/Cas9 基因编辑系统，并测试了两种不同的 gRNA，以分别以 10% 和 35% 的删除效率删除 pyrG 直系同源物。最后，使用潮霉素 B 和 5-氟乳清酸抗性亲本菌株建立了性杂交方案。在选择培养基上杂交和分离后代在一周内完成。为 T. aurantiacus 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。