Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People's Republic of China.
Appl Microbiol Biotechnol. 2023 Mar;107(5-6):1845-1861. doi: 10.1007/s00253-023-12417-3. Epub 2023 Feb 9.
Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.
氮代谢可以调节灵芝的菌丝生长和次生代谢。谷氨酸草酰乙酸转氨酶（GOT）作为细胞内氨基酸代谢的重要酶，在动植物中有许多生理功能，但在真菌中的功能研究较少。本研究在灵芝中发现了两种 GOT 同工酶；一种位于线粒体（GOT1），另一种位于细胞质（GOT2）。在 GOT1 沉默菌株中，活性氧（ROS）水平升高，约为野生型（WT）菌株的 1.5 倍，而沉默 GOT2 则不影响 ROS 水平。为了探讨 GOT 如何影响灵芝中的 ROS，进行了与细胞内 ROS 产生和消除相关的实验。首先，与 WT 菌株相比，当敲低 got1 或 got2 时，GOT 的一种底物谷氨酸的含量降低，而谷胱甘肽（l-γ-谷氨酰-l-半胱氨酸甘氨酸）（GSH）的含量分别降低了约 38.6%、19.3%和 40.1%在 got1 沉默菌株、got2 沉默菌株和 got1/2 共沉默菌株中。其次，GOT 还影响葡萄糖代谢。在 got1 和 got2 沉默菌株中，丙酮酸（PA）、乙酰辅酶 A 和 α-酮戊二酸（α-KG）的含量降低，参与糖酵解途径和三羧酸循环的大多数基因的转录水平增加。在 got1 沉默菌株和 got2 沉默菌株中，NADH 含量增加，NAD/NADH 比值降低，这可能导致线粒体 ROS 的产生。与 WT 菌株相比，got1 沉默菌株中的线粒体 ROS 水平约为 WT 菌株的 1.5 倍。此外，沉默 got1 或 got2 导致抗氧化酶，包括超氧化物歧化酶、过氧化氢酶、谷胱甘肽还原酶和抗坏血酸过氧化物酶的含量降低。最后，与 WT 菌株相比，got1 沉默菌株中的灵芝酸（GA）含量增加了约 40%，而沉默 got2 则导致 GA 生物合成增加了 10%。这些发现为 GOT 对真菌中 ROS 和次生代谢的影响提供了新的见解。
• GOT 在灵芝中的 ROS 水平中发挥重要作用。
• 沉默 got1 导致 GSH 含量和抗氧化酶活性降低，但灵芝中的线粒体 ROS 水平升高。
• 沉默 got1 和 got2 导致灵芝中灵芝酸生物合成增加。
