嗜酸性氧化亚铁硫杆菌亚铁氧化电子传递链的基础研究

The biological metallurgy is an effective technology for the economical and green metal extraction from the intricate, unwieldy and low-grade ores. It accords with the characteristics of mineral resources widespread in China and conforms to the strategy of sustainable development in our country. However, the low bioleaching efficiencies limit its wide application in biomining. The way lies mainly on the improvement of the ferrous oxidation ability of leaching bacteria to solve the low efficiency of bioleaching and expand the application of the technology. Different bioleaching bacteria have evolved diverse ferrous iron oxidizing mechanisms, which remain unclear till now. Our research mainly focuses on the furcated electron transfer model of ferrous iron oxidation, which was put forward in Acidithiobacillus ferrooxidans ATCC 23270, the model strain in bioleaching, by using the gene markerless replacement and overexpression system established in our lab. Firstly, we try to clarify the function of the first electron receptor Cyc2 by knocking out both cyc2 and its substitute gene AFE_1428 in the cyc2 knockout mutant A. ferrooxidans Δcyc2, followed by the testing the activity of ferrous iron oxidation in the double knockout mutant. Secondly, we try to analyze the exact functions of the electron transfer chain and some key electron transfer proteins, which are encoded by the rus and petI operons, by way of gene overexpression in plasmid and integration in the chromosome of A. thiooxidans ATCC 19377, respectively. Finally, the growth properties and ferrous iron oxidizing activities of the mutant, wild type and engineered strains will be detected and compared, as well as the transcription profiles of genes involved in the two operons, in order to analyze the key proteins or the speed limit point in the electron transfer chain. Then these proteins will be further modified to construct the engineering bacteria with improved ferrous iron oxidation capacity. As a result, this study not only helps to elucidate the ferrous iron oxidation mechanism of A. ferrooxidans, but also provides a theoretical and methodological guidance for constructing engineering strains with steady higher activities of ferrous iron oxidation for industrial applications. Therefore, our research has important significance both on academic studies and industrial applications.

生物冶金是经济、绿色开发矿产资源的有效技术，符合我国可持续发展战略。提高浸矿细菌的亚铁氧化能力是解决生物浸矿效率低、扩大实际应用的有效途径。目前对浸矿细菌的亚铁氧化机制研究尚不深入，本项目利用我们建立的遗传操作平台、研究优势和前期研究成果，从分子水平对生物浸矿模式菌嗜酸性氧化亚铁硫杆菌亚铁氧化电子传递链模型进行深入研究和验证。拟通过敲除cyc2和其替代基因AFE_1428，研究传递链第一个电子受体蛋白的功能和双敲除突变株的亚铁氧化能力；通过将rus、petI操纵子上传递体蛋白编码基因，分别以质粒和整合在染色体上两种形式导入嗜酸性氧化硫硫杆菌中异源表达，研究亚铁氧化电子传递链及其传递体蛋白的功能；通过分析明确传递链中关键传递体蛋白和限速位点，进一步构建亚铁氧化能力提高的工程菌。本研究不仅可以从分子水平揭示该菌的亚铁氧化机制，也可为构建高效亚铁氧化工程菌、提高浸矿效率提供理论依据。

生物冶金符合我国矿产资源特点和我国可持续发展战略，但是浸矿细菌的亚铁氧化效率低，限制了这项技术大规模的工业应用。深入研究浸矿细菌的亚铁氧化机制，构建高效亚铁氧化工程菌是解决生物浸矿效率低问题的有效途径。目前对浸矿细菌的亚铁氧化机制研究尚不深入，本项目以生物冶金优势菌嗜酸性氧化亚铁硫杆菌的亚铁氧化电子传递链模型为研究对象，首先对具有亚铁氧化能力的A. ferrooxidans ATCC 23270 和不具有亚铁氧化能力的A. thiooxidans ATCC 19377 进行了比较基因组学分析，确定编码亚铁氧化电子传递链的petI和rus操纵子是A. ferrooxidans氧化亚铁的特有基因；接着利用本课题组在极端嗜酸性硫杆菌中建立的遗传操作平台和研究优势，通过在A. ferrooxidans中构建基因过表达菌株，研究和比较了rus操纵子的cyc2、cyc1、cup、coxABCD、rus基因在该菌亚铁氧化中的功能，明确了电子传递链上的关键位点；通过基因敲除、基因过表达及与Rus蛋白的体外互作，证明电子传递链第一个电子受体蛋白编码基因cyc2和其功能替代基因AFE_1428在该菌亚铁氧化中都发挥重要作用；将编码电子传递链蛋白的petI和rus操纵子分别导入A. ferrooxidans中过表达和导入A. thiooxidans中异源表达进行了功能验证，研究结果为理论上揭示嗜酸性氧化亚铁硫杆菌亚铁氧化机制提供了实验数据，为应用上提高该菌亚铁氧化能力的遗传改造奠定了基础，具有理论和实际应用双重重要意义。