嗜酸性氧化亚铁硫杆菌铜抗性机制的基础研究与应用

The bioleaching technology, which accords with the characteristics of mineral resources and the strategy of sustainable development in our country, has been widely applied to extract metal ions from minerals. However, the bioleaching efficiencies are influenced by many factors, one of which is the high concentration of heavy metal ions. For the deficiency of efficient genetic manipulation systems in bioleaching microorganisms, the heavy metal tolerance mechanisms of those microorganisms remain unclear until now. In this study, Acidithiobacillus ferrooxidans ATCC 23270, an important model strain in bioleaching, is used to elucidate the copper homeostatic mechanism by using the markerless gene replacement system established in our lab recently. Our research mainly focuses on the following three aspects. Firstly, to identify the relevant copper tolerance genes by RNA sequencing and RT-qPCR experiments, then subject the obtained genes to functional cluster analysis. Secondly, to analyze the exact functions of some key genes, which are involved in copper tolerance, and that of the Cop and PolyP-Pho84 system in the copper tolerance in A. ferrooxidans ATCC 23270 by way of gene knockout and overexpression, and then depict a copper tolerance model in this strain. Finally, to construct higher copper tolerance A. ferrooxidans strains for industrial bioleaching by integrating the pivotal copper tolerance genes onto its chromosome. As a result, our study will not only provide experimental data for elucidating the copper homeostatic mechanism of A. ferrooxidans, but also provide theoretical and methodological guidance for studies of the copper homeostatic mechanisms of other bioleaching microorganisms and of the other heavy metal homeostatic mechanisms. Moreover, our study will be helpful to construct engineering strains with steady higher copper tolerance, and finally lay foundations for improving the bioleaching efficiency.

生物冶金符合我国矿产资源的特点和可持续发展战略，但生物浸出效率受很多环境因素影响，其中浸出体系中高浓度重金属离子是重要限制因素之一。由于浸矿微生物缺乏完善的遗传操作体系，目前对其重金属抗性机制研究尚不深入。本项目以生物浸出模式菌嗜酸性氧化亚铁硫杆菌(A.f)为研究对象，使用我们建立的遗传操作体系，对其铜抗性机制展开研究。首先通过转录组测序和RT-qPCR验证，分析确定A.f铜抗性相关基因，并进行生物学功能聚类分析；其次，通过基因敲除和过表达研究，分析关键铜抗性基因和Cop、PolyP-Pho84系统在该菌铜抗性中的作用，揭示其铜抗性机制；最后，通过将关键铜抗性基因整合到A.f染色体上构建高效抗铜菌株。本研究可为阐明A.f铜抗性机制提供实验数据，并为其它浸矿微生物铜抗性乃至其它重金属抗性研究提供理论和方法指导；也利于构建稳定遗传的高效抗铜基因工程菌，最终为提高生物浸出效率奠定基础。

生物冶金符合我国矿产资源特点，尤其适用于铜矿的开采，但是浸出效率受浸出体系中高浓度铜离子的制约，研究浸矿微生物的铜抗性机制，构建高效抗铜基因工程菌是解决问题的有效途径。嗜酸性氧化亚铁硫杆菌是生物冶金的优势菌，也是目前报道抗铜能力最强的浸矿细菌，但因其遗传操作难度大，对其铜抗性机制研究尚不深入。本项目首先通过转录组测序和RT-qPCR验证，筛选分别以亚铁和硫为能源时与该菌铜抗性相关的差异表达基因，构建了该菌铜抗性相关差异表达基因库，并分析了关键铜抗性基因；然后使用本实验室建立的A. ferrooxidans基因无痕敲除方法，对该菌Cop、polyP抗铜系统的copA1、copB和ppx基因进行了敲除，获得相应敲除突变株，通过检测敲除突变株在铜离子刺激下的生长特性和铜抗性基因的表达差异，分析了各敲除基因及Cop、polyP系统在该菌铜抗性中的作用；同时将该菌Cop、polyP抗铜系统的copA1、copB和ppx、ppk1、ppk2基因分别与强启动子Ptac连接，通过广泛宿主质粒pJRD215导入A. ferrooxidans野生型菌株中，构建了基因过表达工程菌，检测了基因过表达工程菌铜离子刺激下的生长特性和铜抗性基因的表达差异，不仅为分析Cop、polyP系统及其相关基因在该菌铜抗性中的作用提供实验数据，也获得了铜抗性明显提高的工程菌，今后可将铜抗性基因在基因组上倍增，构建遗传稳定的高效抗铜菌株用于工业应用；此外，还选取对铜离子刺激响应明显的A. ferrooxidans基因的启动子与gusA报告基因构建表达盒，探索了构建A. ferrooxidans铜离子生物传感器的可行性。本项目研究结果为阐明A. ferrooxidans铜抗性机制提供了实验数据，为进一步有目的的遗传改造构建高效抗铜基因工程菌奠定了基础，具有理论和实际应用双重重要意义。

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