Liu Dandan, Liu Qian, Guo Wenzhu, Liu Yin, Wu Min, Zhang Yongli, Li Jingen, Sun Wenliang, Wang Xingji, He Qun, Tian Chaoguang
State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Biology (Basel). 2022 Sep 24;11(10):1396. doi: 10.3390/biology11101396.
The filamentous fungus is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type , the transformation of industrially used strains with few conidia (e.g., N1) or that are even aconidial (e.g., O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and -mediated transformation of the strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The -mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 deletion mutants were constructed in the N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of .
丝状真菌被发酵工业广泛用于生产酶，尤其是葡糖淀粉酶。尽管多种遗传技术已成功应用于野生型菌株，但对分生孢子较少（如N1）甚至无分生孢子（如O1）的工业用菌株进行转化仍然很费力。在此，我们开发了遗传工具，包括原生质体介导的转化以及使用绿色荧光蛋白作为报告标记对N1和O1菌株进行介导的转化。在优化了从菌丝体释放原生质体的各种因素后，原生质体介导的转化效率对于N1达到89.3%（25/28），对于O1达到82.1%（32/39）。介导的转化效率对于N1为98.2%（55/56），对于O1为43.8%（28/64）。我们还开发了一种无标记的CRISPR/Cas9基因组编辑系统，使用基于AMA1的质粒来表达Cas9蛋白和sgRNA。在22个转化体中，使用此处开发的原生质体介导的转化方法和无标记的CRISPR/Cas9系统在N1背景下构建了9个缺失突变体。在此改进的基因组编辑方法将加速对这些工业真菌中葡糖淀粉酶高产机制的阐明，并将有助于在其他工业菌株中使用高效的靶向突变。
