Abstract Roles of functional genes in secondary metabolism of mushroom-forming basidiomycetes are rarely clarified due to the difficulty of gene knock-out in them. In Ganoderma lucidum, an antitumor ganoderic acid (GA) producing basidiomycete, two homologs of a Cys2-His2 (C2H2)-type zinc finger transcription factor (CRZ1) - GlCRZ1 and GlCRZ2 were found by bioinformatic analysis. Using a recently developed CRISPR/Cas9 genome editing platform, each gene of the two CRZ1 orthologs (glcrz1 and glcrz2) was disrupted to reveal their function in calcium signaling of GA biosynthesis. Compared to the wild type strain (WT), in which Ca2+ addition enhanced the GA production, disruption of glcrz1 did not hamper the cell growth but abolished the calcium signaling effect on stimulating GA biosynthesis. For the glcrz2 knocked-out strain, both the mycelial growth and GA synthesis were severely attenuated and its GA production declined even with Ca2+ treatment. These results indicated that the calcium signaling via GlCRZ1 and GlCRZ2 were critical in the calcineurin-mediated regulation of GA biosynthesis by G. lucidum. The work may be also helpful to understand the function of CRZ1 in secondary metabolism regulation in other basidiomycetes.

中文翻译：

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钙调神经磷酸酶响应转录因子CRZ1在灵芝生物合成灵芝酸中的作用

摘要 由于基因敲除难度较大，功能基因在成蘑菇担子菌次级代谢中的作用鲜有阐明。在灵芝，一种抗肿瘤灵芝酸（GA）产生担子菌中，通过生物信息学分析发现了Cys2-His2（C2H2）型锌指转录因子（CRZ1）的两个同源物——GlCRZ1和GlCRZ2。使用最近开发的 CRISPR/Cas9 基因组编辑平台，破坏了两个 CRZ1 直向同源物（glcrz1 和 glcrz2）的每个基因，以揭示它们在 GA 生物合成的钙信号传导中的功能。与野生型菌株 (WT) 相比，其中 Ca2+ 添加增强了 GA 生产，glcrz1 的破坏不会阻碍细胞生长，但消除了钙信号对刺激 GA 生物合成的影响。对于 glcrz2 敲除菌株，菌丝生长和 GA 合成都严重减弱，即使采用 Ca2+ 处理，其 GA 产量也下降。这些结果表明，通过 GlCRZ1 和 GlCRZ2 的钙信号传导对于灵芝 GA 生物合成的钙调神经磷酸酶介导的调节至关重要。这项工作也可能有助于了解 CRZ1 在其他担子菌的次级代谢调节中的功能。