耐铬菌株中还原酶介导环境铬(VI)降解及其诱导小鼠肝细胞功能的机制研究

Heavy metal pollution threatens ecosystem and public health in China and abroad. Hexavalent chromium （Cr(VI)）-induced digestive damage and health risks caused by water and food exposure has arisen increasingly concern. It is urgent to find effective and safe methods to remove Cr(VI). Bioremediation method has been widely considered to be an economic and environmentally benign strategy for Cr(VI) detoxification. However, there should be a clear understanding of the role of Cr(VI)-resistant genes in the efficient degradation strains prior to their application in contaminated environments. A novel Cr(VI)-degrading bacterium Sporosarcina saromensis M52 was isolated from the intertidal zones in Maluan Bay, Xiamen and reported for the first time by us. The bioremediation methods and molecular-biological mechanisms of M52 will be investigated by Cr(VI)-degrading genes screening, genetically engineered bacterium construction and degradation effects research. The hepatotoxicity during degradation process will also be studied in vivo. This study will deepen the knowledge of the bioremediation mechanisms of Cr(VI)-resistant bacterium. In addition, it can provide the theoretical basis and technical supports for Cr(VI) pollution by bioremediation method, evaluate its environmental and biological safety and is meaningful for reducing population exposure to Cr(VI).

重金属污染严重威胁我国生态安全和公众健康，人群经饮水和饮食暴露于六价铬引起的消化系统损伤和健康风险备受关注，安全有效去除环境中的六价铬污染成为亟待解决的环境科学难题。生物修复是一种新型的污染处理技术，发现高效降解菌株、阐明降解机制对于生物修复环境六价铬污染至关重要，其中还原酶介导六价铬降解的分子机制尚不清晰。我们在分离获得一株马銮湾土著耐铬菌株Sporosarcina saromensis M52的基础上，拟研究M52对六价铬的降解途径及降解机制；筛选参与降解的六价铬还原酶及其功能基因（簇），并对功能基因进行克隆、重组表达，构建高效表达六价铬还原酶的基因工程菌；优化该工程菌的降解条件，并从动物水平，研究工程菌处理六价铬后降解体系的肝脏毒性，分析应用前景。本项目的完成将阐明还原酶介导六价铬降解的分子机制，为生物防治六价铬污染提供理论和技术支持，对改善生态环境、减少人群六价铬暴露具有重要意义。

①明确了菌株M52耐受及去除Cr(VI)的主要途径为将Cr(VI)还原为Cr(III)，而非吸附，且受温度和pH的交互作用影响，Cu2+和Fe2+促进M52对Cr(Ⅵ)的去除，SDS和Triton X-100抑制M52对Cr(Ⅵ)的去除。②筛选并获得orf0415、orf2987、orf3458等多个参与菌株M52耐受与还原Cr(Ⅵ)的相关功能基因，并在此基础上成功构建表达上述基因的重组菌株，证明其环境适应性优于原始菌株M52，提示重组菌株在修复复杂环境Cr污染或多种重金属污染中的广泛前景。③初步阐明M52耐受及去除Cr(VI)的分子机制包括：编码表达Cr(Ⅵ)还原酶，减少胞质内外Cr(Ⅵ)浓度；增强主动外排Cr(Ⅵ)，降低Cr(Ⅵ)对菌体的直接损伤；编码表达具有DNA修复能力的蛋白酶，减轻ROS对遗传物质的氧化性损伤；增加鞭毛合成相关蛋白的表达，增强运动性以趋避高浓度Cr(Ⅵ)；激活Tat途径，确保蛋白质的正确合成；通过群体感应协同抵抗Cr(Ⅵ)胁迫；菌株通过表面基团及功能键的改变，参与Cr(VI)的去除；Cr(VI)可在细菌表面被还原为Cr(III)，菌株表面的氨基、酰胺基、醇基、羧基等官能团可为Cr(VI)的还原提供电子等；提出ABC transporters, Two-component system等通路值得进一步深入探究。④在动物水平上，Cr(VI)经原始菌株M52和重组菌株处理后，未见对小鼠肝脏的明显毒性损伤，在一定程度上打消了菌株用于Cr(VI)污染修复时是否会导致健康损伤的安全顾虑。本项目的完成不仅明确了菌株M52去除Cr(VI)的还原途径，从分子和细胞水平揭示了Cr还原酶参与耐受与去除Cr(VI)的机制，证明了重组菌株优于原始菌株的环境适应能力，为借助基因工程手段开展生物修复环境Cr(VI)污染提供了理论和技术支持；在动物水平开展的菌株去除Cr(VI)前后的健康效应研究，更为微生物制剂用于环境污染的安全性评价提供了参考，对高效、安全去除环境中的重金属污染、改善生态环境、保护人群健康具有重要意义。