专性化能自养嗜酸性氧化亚铁硫杆菌亚铁氧化和硫氧化代谢的基础研究

Bioleaching is an effective technology for metal extraction from the low-grade, intricate and unwieldy ores widespread in China. It has great advantages, such as low cost, less investment and clean process. It is also in accordance with our national strategy of sustainable development. Experimental researches indicate that the crux of bioleaching efficiency lies on the microbial oxidizing abilities of ferrous iron and reduced inorganic sulfur compounds. Compared with traditional domestication and mutagenic breeding methods, the genetic engineering approach is more effective in exploring the microbial ferrous iron- and sulfur-oxidizing mechanisms and becoming an important research field on the improvement of bioleaching microorganisms. Our work focuses on the model bioleaching microorganism Acidithiobacillus ferrooxidans. Based on our technical advantage of the genetic manipulation, the key genes involved in ferrous iron- and sulfur-oxidizing pathways will be knocked out and over-expressed in A. ferrooxidans ATCC 23270. The growth properties and transcription profiles of the wild-type, mutant and engineered strains will be detected and compared, as well as their abilities of ferrous iron- and sulfur-oxidation, to analyze and confirm the function of the key enzymes involved in the energetic metabolic pathways of A. ferrooxidans. Our study can not only provide experimental data for the existing models of ferrous iron- and sulfur-oxidizing metabolic pathways in A. ferrooxidans ATCC 23270, which are merely set up based on bioinformatic predictions and genomic, transcriptomic, or proteomic analyses, but also be helpful for the perfectness of the models. On the other hand, our results can help us to obtain more knowledge on the regulation mechanism of the energetic metabolic pathways in A. ferrooxidans, and to find out the rate-limiting steps in the global network for further improvement. As results, the engineered A. ferrooxidans strains with effectively improved ferrous iron- and sulfur-oxidizing abilities will be obtained for application in bioleaching industry. So, our work has important significance both on academic studies and applications.

生物冶金符合我国矿产资源的特点和可持续发展战略，提高浸矿微生物亚铁和硫氧化能力是提高生物冶金浸矿效率的关键。传统的人工驯化和诱变育种不能从根本上解决问题，深入探究浸矿微生物亚铁和硫氧化机制，通过基因工程手段进行遗传改造正成为研究的焦点。本项目以浸矿模式菌嗜酸性氧化亚铁硫杆菌(A.f)为研究对象，结合自己的研究优势和建立的遗传操作平台，在A.f中敲除和高效表达其亚铁和硫氧化途径的关键酶基因，通过比较检测不同基质培养的突变株和工程菌的生长状况、亚铁和硫氧化能力、关键酶基因转录水平等，分析确定A.f能量代谢途径关键酶的功能。实验结果不仅可为基于生物信息学和组学推测的亚铁和硫氧化代谢模型提供实验数据，有助于模型的完善，而且可加深对A.f能量代谢调控的了解和认识，针对限速靶点进行遗传改造，提高A.f亚铁和硫氧化代谢能力，为工业生产构建高效浸矿工程菌。本项目具有理论研究和实际应用双重重要意义。

在生物冶金中，浸矿细菌的亚铁和硫氧化能力与生物冶金效率密切相关。本项目以浸矿模式菌株嗜酸性氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans ATCC 23270）为研究对象，以提高其亚铁和硫氧化能力为目的，探究了该类自养细菌亚铁氧化和硫氧化代谢途径中某些关键酶的功能。取得的主要研究结果：借助本实验室在基因敲除和转基因方面的研究优势，分别选取硫氧化代谢途径中的sdo, tetH, doxDA操纵子, hdr操纵子和亚铁氧化代谢相关的cyc2 和rus，通过构建基因敲除突变株和基因过表达工程菌，比较检测了基因的敲除和过表达对菌的生长状况、亚铁和硫氧化能力、关键酶基因转录水平的影响，为分析A. ferrooxidans亚铁氧化和硫氧化途径中这些关键酶的功能提供了实验数据，加深了对A. ferrooxidans亚铁和硫氧化代谢的认识。同时，在实验技术方面，进一步优化了无标记基因敲除方法，并且在A. ferrooxidans中建立了基于β-葡萄糖苷酸酶的报告基因系统，使这类极端嗜酸性自养细菌的遗传操作更便利。本项目获得了预期研究结果，达到了预期研究目标，具有理论研究和实际应用双重重要意义。