Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan.
Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan.
Appl Microbiol Biotechnol. 2021 Oct;105(20):7813-7823. doi: 10.1007/s00253-021-11595-2. Epub 2021 Sep 24.
This study focuses on sirtuins, which catalyze the reaction of NAD-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5 mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5 mM hydroxyurea and 50 µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.
本研究聚焦于烟酰胺腺嘌呤二核苷酸（NAD）依赖性蛋白去乙酰化酶反应的去乙酰基酶 Sirtuins，以提高棉子实雷氏酵母（Ashbya gossypii）中的核黄素产量。烟酰胺是 Sirtuins 的已知抑制剂，在 5 mM 时，它会使棉子实雷氏酵母菌落的颜色呈现更深的黄色。棉子实雷氏酵母有 4 个 Sirtuins 基因（AgHST1、AgHST2、AgHST3、AgHST4），我们对其进行了破坏，以研究 Sirtuins 在棉子实雷氏酵母核黄素生产中的作用。获得了 AgHST1∆、AgHST3∆和 AgHST4∆菌株，但未获得 AgHST2∆。AgHST1∆和 AgHST3∆菌株产生的核黄素量比 WT 菌株分别高出约 4.3 倍和 2.9 倍。AgHST3∆菌株的人源 Sirtuin 6（SIRT6）样活性低于 WT 菌株，只有在 AgHST3∆菌株中，组蛋白 H3 K9 和 K56 的乙酰化（H3K9ac 和 H3K56ac）水平明显高于 WT 菌株。这些结果表明，AgHst3 是棉子实雷氏酵母中的 SIRT6 样 Sirtuins，其活性会影响棉子实雷氏酵母的核黄素产量。在 5 mM 羟基脲和 50 µM 喜树碱（可引起 DNA 损伤，尤其是双链 DNA 断裂）存在的情况下，WT 菌株的菌落颜色变成更深的黄色。此外，羟基脲显著导致核黄素产量增加约 1.5 倍，而喜树碱也增强了核黄素的生产，尽管未检测到显著差异。喜树碱倾向于增加 H3K56ac 的含量，但羟基脲处理并未增加 H3K56ac 的含量。本研究表明，AgHst1 和 AgHst3 通过 NAD 代谢和 H3 的乙酰化分别参与棉子实雷氏酵母的核黄素生产。这一新发现是阐明 Sirtuins 在棉子实雷氏酵母核黄素过量生产中的作用的重要一步。
关键点
烟酰胺增强了棉子实雷氏酵母的核黄素产量。
破坏 AgHST1 或 AgHST3 基因也会增强棉子实雷氏酵母的核黄素产量。
H3K56 的乙酰化导致棉子实雷氏酵母核黄素产量的增加。
