基于芽孢杆菌细胞重编程的重金属捕捉机理研究

Biosorption of heavy metals (HM) by bacteria has attracted more and more concern due to the low cost and high efficiency. However, the large amount of excess activated sludge produced by biosorption treatment tends to cause the secondary pollution because of their high content of heavy metals. To solve this problem, and meanwhile improve the biosorption efficiency, we aim to establish a Riboswitch-based heavy metal sensing system and thereby reprogram Bacillus strains to detect the heavy metal ions and precipitate them by secreting heavy metal biosorbent, and after that initiate self-lysis to control the production of activated sludge. Our proposed work is based on our recent work on the establishment of the Bacillus genome editing system, as well as the discoveries that the PHB nanoparticle-displayed long metallothioneins chelate and precipitate heavy metals with high efficiency. The key issues for this project are the design of heavy metal biosensing system, and the fine control of the heavy metal capture system and cell lysis system by the heavy metal recognition signals. We will employ the genome editing technique that we have recently developed to generate the random mutation library of the hammerhead ribozyme, in order to screen for heavy metal ion specific riboswitches. The reporter genes, spectrometry, and computer aided design will be applied to moniter the expression model of the heavy metal sensing program, heavy metal capture program, as well as the self-lysis program, thereby optimize the signal transduction pathways among them. The results will provide new thoughts and approaches to the design of artificial riboswitches as well as the regulation of cell function.

微生物吸附技术在污水重金属处理方面具有广阔的前景，但剩余污泥中细胞积累的重金属难以回收极易造成二次污染，如何控制剩余污泥并有效回收重金属已成为亟待解决的问题。本项目立足于前期工作中对PHB-MT（聚羟基丁酸酯-金属硫蛋白）纳米生物材料螯合沉淀重金属离子的研究，提出利用细菌分泌的纳米颗粒捕捉重金属的策略，通过细胞编程使菌体完成重金属捕捉功能后发生自裂解，从源头控制剩余污泥的产生。本研究拟采用独创的染色体编辑系统构建锤头状核酶第二茎环的随机突变库，利用抗性基因筛选特异性结合重金属离子的核糖开关(Riboswitch)用以重编程芽孢杆菌，使其灵敏感应重金属离子并相应分泌出PHB-MT螯合沉淀重金属离子，在沉淀完毕后开启细胞凋亡程序。进一步利用温敏型内含肽控制PHB与MT的分离，通过pH梯度洗脱回收与MT结合的重金属离子。本项目将为人工核糖开关研究与细胞功能调控提供新的思路。

微生物吸附技术在污水重金属处理方面具有广阔的前景，但剩余污泥中细胞积累的重金属难以回收极易造成二次污染，如何控制剩余污泥并有效回收重金属已成为亟待解决的问题。本项目利用细菌分泌的纳米颗粒捕捉重金属，通过细胞编程使菌体完成重金属捕捉功能后发生自裂解，从源头控制剩余污泥的产生。项目开展过程中，成功筛选出高效金属硫蛋白MT3和响应镉离子的核酸适体HMR-6，并通过染色体编辑技术构建了工程菌株Bacillus subtilis HMC9，在复杂污水环境中灵敏识别重金属离子,并分泌捕捉剂将其沉淀；重金属清除后菌株自我裂解,以控制剩余活性污泥的产生；沉淀物经温度和pH调节后可回收具有重要工业价值的聚羟基丁酸酯和重金属,为重金属废水的资源化利用提供了新的生物治理方法。