乳酸乳球菌细胞壁耐酸调控机制及重构

Significant protein expression changes related with cell wall synthesis were demonstrated by previous investigations in Lactococcus lactis F44 characterized by high nisin yield under acid stresses. Meanwhile, the acid tolerance response and the nisin production capability were improved with the over-expression of genes related with cell wall synthesis. However, regulatory mechanisms of the cell wall synthesis remain unclear and need to further research under acid responses. Therefore, in this project, we attempt to obtain the regulator proteins related to cell wall synthesis on the acid response and determine the function and other targets of these proteins. Furthermore, we aim to understand the regulating mechanisms of the cell wall synthesis better and construct a regulatory network preliminarily. Our studies could improve the acid tolerance by efficient expression system of cell wall synthesis relating genes with synthetic biology method and enhance the adaptability of the strains during the industrial production. This project will help to understand regulatory mechanisms of the cell wall synthesis under acid responses, it also provide research basis for studing acid tolerance mechanism of L. lactis cell wall.

前期工作表明，高产乳酸链球菌肽（nisin）的乳酸乳球菌F44在酸性条件下，细胞壁合成相关蛋白表达量出现显著变化；同时，系统的高表达细胞壁合成相关基因也会提高菌株的耐酸能力和nisin的生产能力。然而，酸应激下乳酸乳球菌中细胞壁合成相关的调控机制研究还不够深入。项目拟调取并鉴定酸性条件下细胞壁合成关键功能基因（氨基酸连接酶、氨基转移酶、糖基转移酶、肽聚糖水解酶等）启动子区的特异结合蛋白，进一步确定其功能和其他作用靶点，总结可能的调控机制，尝试研究酸性条件下细胞壁合成调控规律，初步构建调控网络。同时，应用合成生物学技术实现在不同酸性条件下启动细胞壁合成高表达系统，提高菌株在生产过程中的适应能力,有利于工业化生产。本项目的研究有助于深入理解酸性条件下乳酸乳球菌细胞壁合成的调控机制，也为进一步研究乳酸乳球菌细胞壁的耐酸机制奠定了基础。

细胞壁是保护细菌的第一道屏障，细菌在面对环境压力会启动多种调节机制。乳酸乳球菌广泛应用于食品工业，并在医药领域具有巨大的应用潜力。目前乳酸乳球菌细胞壁生物合成的调控机制尚不清楚。本课题鉴定了酸胁迫下细胞壁合成功能基因murD、murE和dacB以及磷壁酸修饰dlt操纵子启动子区的特异结合蛋白，结合蛋白组学、ChIP-seq、EMSA等方法分析找到CodY、TcsR7和多个潜在的转录调控因子，进一步结合组学和荧光定量PCR等技术研究其功能和其他作用靶点，全面总结了转录因子CodY调控机制及酸胁迫下TcsR7对细胞壁磷壁酸调控机制。同时，研究了O-乙酰化和N-去乙酰化修饰对细胞壁肽聚糖结构影响，结果表明O-Ac-M和N-deAc-G修饰能够促进细胞壁的刚性结构的保持，进而促进菌体酸耐受能力的提高以及nisin产量的提升。研究发现转肽酶RacD通过感受pH变化来调控细胞壁肽聚糖含量变化。此外，基于转录组深度测序发现8个耐酸相关小RNA，包括s015、s042、s263、c277等多个sRNA。深入分析发现s263可通过与murA、murC、mraY的RBS区结合，促进细胞壁合成，为细胞壁合成转录后调控机制提供了新的理论指导。本研究分别从转录水平、转录后调控、乙酰化修饰等方面对细胞壁调控进行全面研究，为细胞壁调控网络的构建奠定了基础。