巴氏醋酸杆菌酸胁迫下膜转运蛋白生理应答机制研究

The feedback inhibition of highly concentrated acetic acid on cell growth and acid production seems a bottleneck in its fermentation. Using Acetobacter pasteurianus Huniang 1.01 as a model strain,this project focuses on a series study on membrane transporter- the key unit in acid resistance. By comparison of the starting strain, acid-resistant strains from adaptive evolution, and highly resistant strain at cell physiological, trancriptional and proteomic level, the mechanisms of acid resistance responsing would be illustrated. The key membrane proteins and its evolution mechanism were calrified by analyzing membrane proteome in different physiological states and on different evolutionary levels. On the above basis, physiological and transcriptional analysis of the mutants which constructed using gene knock-out and over-expression strategy were done. From the above results, the relationship of physiological state - key membrane transporter - evolution mechanism -response regulator mechanism was described and it would integrated to extensively explain the regulatory roles and molecular evolution mechanisms of those key membrane transporters under acid stress.This project is expected to provide not only a theoretical reference for further rational breeding of A. pasteurianus, the mechanism to be illustrated might also have its vulue on guiding the development of methodology and theory on the related issues of other acid producing bacteria.

高浓醋酸对产生菌生长和产物合成的反馈抑制作用是制约醋酸发酵的关键问题。本课题以醋酸杆菌Acetobacter pasteurianus 沪酿 1.01 为模式菌株，针对膜转运蛋白这一抵抗酸胁迫的关键单元，通过比较出发菌株、适应性进化获得耐酸菌株和高耐受株，从细胞生理、转录和蛋白组水平分析其生理应答机制。首先分析不同生理状态和不同进化层次下的醋酸杆菌膜转运蛋白差异，确定耐酸关键膜蛋白并解析其进化分子机制。在此基础上，通过基因敲除和过量表达验证相关蛋白并构建突变株，对其进行生理和转录分析。从生理状态－关键膜转运蛋白－进化分子机制－应答调控的关联机制角度阐明关键膜转运系统在A. pasteurianus 抵御酸胁迫环境的生理应答机制。本研究不仅可为进一步理性选育A. pasteurianus提供理论支持，还将为其它产酸细菌的耐酸机制的解析提供理论和方法学上的指导。

本课题以醋酸杆菌Acetobacter pasteurianus 沪酿 1.01 为模式菌株，经过三年研究，通过比较出发菌株、适应性进化获得耐酸菌株和高耐受株，从细胞生理、基因和转录水平解析了适应性进化下的生理应答机制。1）醋酸胁迫处理与紫外诱变相结合筛选得到一株高产菌，通过重复补料分批发酵，确定了PQQ-ALDH为限制性酶，其遵循ordered Bi-Bi机制。在动力学研究中引入“氧气消耗对醋酸积累的化学计量系数”，醋酸转化率达到93.36%，生产强度至 1.77g/L*h。2) 对A. pasteurianus CICIM B7003进行基因和转录分析，发现在高酸浓度下，代谢通路中TCA循环相关酶转录水平均得到增强；膜上氧化通路中，adh、aldh、nadh和辅酶代谢相关的四个基因得到了增强。耐酸直接相关元器件中，ABC转运子无明显变化，F1F0 ATPas相关两个基因转录水平有显著增强，内部消化和辅基也有适应性变。3）研究比较了A. pasteurianus ATCC33445, CICIM B7003-01和CICIM B7003-02 在适应性进化中细胞形态、生理、基因转录及能量调节方面的响应。发现全过程呼吸链上的adh、cyt bd、cyt o和fapA基因转录水平始终上升，确定了能量是耐酸的关键核心。高酸下菌体adh和aldh基因表达的动态调整维持胞内较低醋酸浓度。呼吸链上其他外排相关酶转录水平提升加快释放能量。4）进一步对突变/对照株的能量代谢分析得知，主发酵阶段，乙醇呼吸链为主要供能代谢途径，其他阶段主要通过苹果酸/琥珀酸回补偶联有氧和底物磷酸化途径产能。以能量代谢为主线对CICIM B7003-01适应性进化过程中的基因和转录水平变化，发现在整个进化过程中（50000世代），基因组DNA组氨酸激酶的调控元件有1个IS，质粒的转录调控因子和共聚转移酶TraD等有5个IS。SNP主要涉及膜蛋白、胞内相关代谢蛋白和调控蛋白等，最终发现Acetobacter pasteurianus在酸胁迫下生理应答机制主要和能量代谢密切相关，产酸通路处于核心环节。