黄铜矿浸出中极端酸胁迫下嗜酸氧化硫硫杆菌硫氧化调控机制

The extremely high-acid condition was beneficial to eliminate the passivation behavior of the jarosite in chalcopyrite bioleaching. Acidithiobacillus sp. can obtain energy from sulfur oxidation and produce large amounts of H+, which are closely related to extreme acid environments. Acidithiobacillus thiooxidans, being directly acid-adapted by a long term, showed a stronger physiological characteristic in resisting acid stress (～pH0.2). However, the internal control mechanism is still not understood. In this research, three strains of A. thiooxidans from different stages of evolution are used as objects. With the assistance of GC-MS, patch clamp and fluorescent double staining tools, the membrane lipid composition, membrane current voltage, hydrogen transfer energy level, and ion channel structure are analyzed, which help reveal the response mechanism to acid stress. The changes of cytokines and microenvironment energy cofactors, electron mediators, amino acid metabolism and stress response factors are compared to reveal the related mechanism of cytoplasmic microenvironment under extreme acid stress. With the analysis of transcriptomics, proteomics and gel retardation adoption, the key fundamental protein is explored and its regulatory model for regulating the key enzyme target gene (smg) in sulfur-oxidization is identified. Based on the above studies, the physiological response mechanism in resisting acid stress will be illustrated from the multi-angels of membrane physiology-intracellular physiology-trans-acting factor-sulfur oxidation mode-environmental adaptation in extremely acid stress. The project will provide a scientific basis for directly modifying bioleaching strain with the high ability in resisting environmental stress.

黄铜矿浸出的极端强酸环境有助于消除黄钾铁矾钝化行为。嗜酸硫杆菌可利用硫氧化供给能量并产生大量H+，与极端酸环境紧密相关。课题组长期耐酸定向驯化的嗜酸氧化硫硫杆菌，呈现优良耐受极端酸胁迫生理特性（～pH0.2），然而对其内在调控机制仍缺乏机理性认识。本项目以3株处于不同进化阶段嗜酸氧化硫硫杆菌为研究对象，利用GC-MS、膜片钳和荧光双染技术，分析膜脂组成、膜电流电压、氢传递能量水平、离子通道结构和膜通透性差异，解析细胞膜电生理对酸胁迫的应激机制。比较胞质微环境能量辅因子、电子传递体、氨基酸代谢和应激严谨反应因子等生理参数波动，揭示极端酸胁迫下胞质微环境调控机制。通过转录、蛋白质组学分析和凝胶阻滞技术，鉴定反式作用因子调控硫氧化关键酶靶基因（smg）方式，进而阐明该菌株膜生理-胞质微环境-反式作用因子-硫氧化调控模式-极端酸环境适应性作用机制。本项目可为选育高耐受环境胁迫的浸矿菌提供科学依据。

本项目围绕嗜酸硫杆菌对极端酸胁迫的生理应答机制，分别从极端酸胁迫调控细胞膜电生理特性、胞质微环境表征、全局转录表达以及硫代谢关键因子等多角度展开大量研究工作。主要可归纳如下。（1）借助恒化培养、GC-MS、膜片钳和PI/Hoechst荧光双染等技术，为了维持细胞内微环境pH稳态，细胞通过增强H+-ATPase活性并消耗ATP方式排出胞内多余质子。氨基酸代谢变得更加活跃，如碱性氨基酸天冬氨酸通过代谢消耗质子减轻胞内酸化。细胞膜不饱和脂肪酸，特别是环丙烷含量增加，有助于增加细胞膜致密性，减少外部质子侵入。（2）为深入探究膜生理特质在抵御极端酸胁迫作用机制，借助CRISPR-Cas9双质粒系统，在E. coli BL21(DE3)完成内源性丙烷脂肪酸合酶编码基因cfa基因敲除和敲入，环丙烷脂肪酸Cy-17:0在E. coli WT、Δcfa和Accfa2(WT)细胞膜脂肪酸组成分别占比15.39%、0.00%和0.91%，证实其在维持膜生理稳态重要作用。通过蛋白三维结构预测及与脂质底物分子对接模拟等，确定CFAS2蛋白关键氨基酸残基位点Glu239、His266和Tyr317。（3）基于转录组学解析A. thiooxidans全局基因转录水平，通过GO注释和COG功能模块分类，确定差异表达基因主要涉及双组分系统、硫代谢、中心碳代谢、氨基酸代谢、运动趋化性等细胞活动。其中硫氧化还原酶编码基因sor和硫氧化系统（Sox）相关基因soxABXYZ、sqr等转录水平均显著上调，暗示A. thiooxidans可通过增强硫代谢产生更多能量方式协作抵御酸胁迫。（4）将显著差转录/表达的硫代谢关键蛋白SQR及其突变体的克隆表达与纯化，SQR蛋白N端和C端具有较强疏水性，SQR蛋白跨膜区域分析，基于AlphaFold2对SQR蛋白的建模与结构分析，确定嗜酸硫杆菌的SQR蛋白结构符合二硫键氧化还原酶蛋白家族的结构特征。结合以上研究结果初步阐明嗜酸硫杆菌在膜生理、胞质微环境、关键作用因子及硫代谢等维度应对极端酸压力的关联作用机制，为未来选育高耐受环境胁迫的浸矿菌及工业微生物底盘细胞构建提供科学依据。