Shi Deng-Ke, Zhu Jing, Sun Ze-Hua, Zhang Guang, Liu Rui, Zhang Tian-Jun, Wang Sheng-Li, Ren Ang, Zhao Ming-Wen
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture; Microbiology Department, College of Life Sciences, Nanjing Agricultural University, Nanjing , 210095, Jiangsu, PR China.
Microbiology (Reading). 2017 Oct;163(10):1466-1476. doi: 10.1099/mic.0.000527. Epub 2017 Sep 13.
The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.
交替氧化酶（AOX）构成线粒体呼吸电子传递途径的一个分支，其作用是维持电子通量并减轻活性氧（ROS）的产生。在本文中，在灵芝中鉴定出一个同源AOX基因。灵芝中AOX基因的编码序列包含1038个核苷酸，编码一个39.48 kDa的蛋白质。利用RNA干扰（RNAi）研究AOX在灵芝中的功能，获得了两个沉默菌株（AOXi6和AOXi21），与野生型相比，其交替途径呼吸效率分别显著降低了约60%和50%。与野生型相比，突变菌株AOXi6和AOXi21中灵芝酸（GA）的含量分别显著增加了约42%和44%。在突变菌株AOXi6和AOXi21中还观察到GA生物合成中中间代谢产物含量升高以及相应基因转录水平升高。此外，与野生型相比，AOXi6和AOXi21菌株中的细胞内ROS含量分别显著增加了约1.75倍和1.93倍。此外，添加ROS清除剂N-乙酰-L-半胱氨酸（NAC）可显著降低AOX沉默菌株中的细胞内ROS含量和GA积累。这些结果表明，AOX通过调节细胞内ROS水平影响GA生物合成。我们的研究揭示了AOX在灵芝次生代谢中的重要作用。
