College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800, Jiangsu, People's Republic of China.
Bioresource Institute of Healthy Utilization, Zunyi Medical University, Zunyi, 563000, Guizhou, People's Republic of China.
World J Microbiol Biotechnol. 2024 Sep 20;40(10):328. doi: 10.1007/s11274-024-04142-4.
Trichoderma longibrachiatum UN32 is a well-documented mutant strain known to produce dendrobine-type total alkaloids (DTTAs). It was serendipitously observed that the addition of Co to the medium resulted in a notable enhancement in DTTAs production in the T. longibrachiatum UN32 strain, accompanied by an upregulating effect on the expression of antioxidase-related genes. Hence, the objective of the present work was to ascertain whether ROS (intracellular levels of hydrogen peroxide) induced by Co treatment has a beneficial or detrimental impact on DTTAs biosynthesis. A comparison of the intracellular levels of hydrogen peroxide (HO) and DTTAs treated with CoCl and CHCOOH revealed that CoCl was the optimal inducer for investigating the relationship between ROS formation and DTTAs production. This was due to the observation that ROS formation was reduced by approximately 4% and DTTAs production was increased by 12.55% in comparison to the CHCOOH treatment. The physiological results revealed that the introduction of Co resulted in the oxidative damage and activation of the expression of intracellular superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, it was confirmed that ROS induced by Co was beneficial to DTTAs production by adding exogenous ROS scavengers. The inclusion of all ROS scavengers, including vitamin C, tocopherol, melatonin, mannitol, and sesamol, resulted in a reduction in ROS accumulation and a concomitant decrease in DTTAs production. Specifically, the addition of melatonin at a concentration of 0.4 mg/L demonstrated significant effects, resulting in a 32.53% (P < 0.01) decrease in ROS accumulation and a 45.22% (P < 0.01) reduction in DTTAs production. Subsequently, the timelines of accumulation of intracellular HO and DTTAs content indicated that ROS are also crucial for normal fermentation without CoCl addition. Specifically, the proper HO dose for DTTAs accumulation is between 8.82 and 18.86 μmol/g. The present study offers the initial experimental evidence indicating that CoCl enhance DTTAs production during the culture of T. longibrachiatum UN32 via leading an increase in intracellular ROS, which is conductive to DTTAs production and can be inhibited by the ROS scavengers. Our results provide insights into the mechanistic study of DTTAs biosynthesis.
长枝木霉 UN32 是一种有大量文献记录的突变株，已知能产生石斛碱型总生物碱（DTTAs）。我们偶然发现，向培养基中添加 Co 会显著提高长枝木霉 UN32 菌株中 DTTAs 的产量，同时上调抗氧化酶相关基因的表达。因此，本研究旨在确定 Co 处理诱导的 ROS（细胞内过氧化氢水平）对 DTTAs 生物合成是有益还是有害。通过比较 CoCl 和 CHCOOH 处理的细胞内过氧化氢（HO）和 DTTAs 的水平，发现 CoCl 是研究 ROS 形成与 DTTAs 产量之间关系的最佳诱导剂。这是因为与 CHCOOH 处理相比，ROS 的形成减少了约 4%，而 DTTAs 的产量增加了 12.55%。生理结果表明，引入 Co 会导致细胞内超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）的氧化损伤和表达激活。此外，通过添加外源性 ROS 清除剂证实，Co 诱导的 ROS 有利于 DTTAs 的产生。添加所有 ROS 清除剂，包括维生素 C、生育酚、褪黑素、甘露醇和芝麻酚，会减少 ROS 积累并降低 DTTAs 的产量。具体而言，添加浓度为 0.4 mg/L 的褪黑素会产生显著效果，导致 ROS 积累减少 32.53%（P<0.01），DTTAs 产量降低 45.22%（P<0.01）。随后，细胞内 HO 和 DTTAs 含量的积累时间线表明，没有 CoCl 加入时，ROS 对正常发酵也是至关重要的。具体来说，DTTAs 积累的合适 HO 剂量在 8.82 到 18.86 μmol/g 之间。本研究提供了初步的实验证据，表明 CoCl 通过增加细胞内 ROS 来促进长枝木霉 UN32 培养过程中 DTTAs 的产生，这有利于 DTTAs 的产生，并且可以被 ROS 清除剂抑制。我们的研究结果为 DTTAs 生物合成的机制研究提供了新的见解。
