功能膜微域在七烯甲萘醌合成过程中的作用解析

Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7

摘要:

功能膜微域(functional membrane microdomains, FMMs)是细菌细胞质膜上富含脚手架蛋白和聚异戊二烯类物质的结构域，参与细胞生命活动的多个过程。本研究主要聚焦于揭示FMMs与MK-7之间的相关性，并对MK-7合成进行代谢调控。首先，通过荧光标记初步确定FMMs和MK-7在细胞膜上存在相关性；其次通过分析FMMs破坏前后细胞膜上MK-7含量的变化以及细胞膜有序度的变化情况，明确MK-7是FMMs的聚异戊二烯类关键组分；接下来，采用可视化分析探究MK-7合成过程中部分关键酶的亚细胞定位，并通过FloA将胞内游离的途径酶Fni、IspA、HepT和YuxO定位至FMMs中，进而实现MK-7合成途径的区室化，最终成功获得一株高产MK-7的枯草芽孢杆菌(Bacillus subtilis)菌株BS3AT，摇瓶水平MK-7产量达到300.3 mg/L，3 L发酵罐中MK-7产量为464.2 mg/L。

Abstract:

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.