Dong Yajun, Cui Shixiu, Liu Yanfeng, Li Jianghua, DU Guocheng, Lü Xueqin, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Jun 25;39(6):2215-2230. doi: 10.13345/j.cjb.230111.
Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
主要由支架蛋白和聚异戊二烯类组成的功能性膜微区（FMMs）在细菌的多种细胞生理过程中发挥着重要作用。本研究的目的是确定MK-7与FMMs之间的相关性，然后通过FMMs调控MK-7的生物合成。首先，通过荧光标记确定细胞膜上FMMs与MK-7之间的关系。其次，通过分析破坏FMMs完整性前后细胞膜上MK-7含量的变化以及膜有序性的变化，证明MK-7是FMMs的关键聚异戊二烯类成分。随后，通过可视化分析探索了MK-7合成中一些关键酶的亚细胞定位，细胞内游离途径酶Fni、IspA、HepT和YuxO通过FloA定位于FMMs，实现了MK-7合成途径的区室化。最后，成功获得了高产MK-7的菌株BS3AT。摇瓶中MK-7的产量达到300.3 mg/L，在3 L发酵罐中达到464.2 mg/L。
