基于改造联苯双加氧酶获取高溴代二苯醚降解菌并用于土壤修复的研究

Polybrominated diphenyl ethers (PBDEs) are common pollutants extremely resistant to degradation. PBDEs usually exist in the form of highly brominated congeners. Highly brominated PBDEs cannot be degraded by known microbes, which hampered the development of remediation techniques for PBDEs-contaminated soil. On the basis of the lower brominated PBDEs degrading bacterium Cupriavidus basilensis WS, this project is going to construct a high-throughput screening system against the key enzyme in the degradation pathway, finally lead to the creation of a degradation tool targeting highly brominated PBDEs in soil: 1) By coupling PBDEs degradation with fluorescence signal, construct a high-throughput screening system against PBDEs degrading ability of biphenyl dioxygenase; 2) After several rounds of mutation and screening targeting the BphA1 subunit of BPDO with the aid of high-throughput screening method, obtain the mutated BPDO capable of degrading highly brominated PBDEs; 3) Introduce the mutated BphA1 into C. basilensis WS using CRISPR-Cas9 mediated genome editing, transforming it into a safe and stable recombinant strain; 4) Modeling the remediation of soil contaminated by highly brominated PBDEs by recombinant strain, with the aim of analyzing the degradation ability of the substrate, concentration of residual end products, the stability of recombinant strain in soil, the stability of mutated bphA1, and the variation of other soil microbes. Major improvements have been made and integrated in several sections of this project. The project will make the degradation of highly brominated PBDEs in soil possible, and provide a new solution for remediation of soil contaminated by halogenated organic compounds.

多溴二苯醚是一类极难降解的常见污染物，多以高溴代形式存在。目前已知的微生物均无法有效降解高溴代二苯醚，致使相关土壤修复技术难以成型。本项目将在低溴代二苯醚降解菌基础上，对其关键酶加以改造，最终获得安全有效的土壤高溴代二苯醚降解工具：1）基于荧光信号偶联，建立针对联苯双加氧酶氧化高溴代二苯醚能力的高通量筛选体系；2）结合高通量筛选方法，对联苯双加氧酶的BphA1亚基进行多轮突变及筛选，获得可转化高溴代二苯醚的改造酶；3）采用基于Cas9的基因组编辑，将BphA1突变引入原始菌，获得安全、稳定的重组菌；4）模拟重组菌修复受高溴代二苯醚污染土壤的过程，考察其底物降解能力及产物残留情况，分析重组菌在土壤中存活能力、突变bphA1基因的稳定性、其他微生物丰度变化等指标。本项目在多个环节进行了技术创新，并将其有机整合，使土壤中高溴代二苯醚的降解成为可能，为受卤代有机物污染土壤的修复提供了新的思路。

目前已知的微生物均无法降解高溴代的PBDEs，导致相关的修复技术难以成型。本研究对低溴代二苯醚降解菌中的关键酶BPDO进行改造后构建了重组菌株，并分析重组菌对高溴代PBDEs的降解行为。结果发现：(1) 通过对BPDO中的bphA1进行突变、筛选，最终可获得具氧化9个取代溴PBDEs的突变体；(2) BPDO突变子对八溴、九溴等高溴代PBDEs的氧化降解率可达80 %以上，且溴代取代基的数量和结构会影响加氧位置，溴取代基越少的苯环更容易发生开环反应，且相对于间/对位取代，活性氧自由基更倾向于攻击二苯醚苯环上的邻位取代；(3) 基因改造后重组菌株的相关降解酶能发挥正常功能，实现对高溴代PBDEs的降解；(4) 重组菌株在土壤中仍具备对高溴代PBDEs的降解能力；(5) 突变基因bphA1在菌中具较高的稳定性，且重组菌株在土壤中具良好的存活情况，也不会对土著微生物造成显著影响。本研究从分子水平对多个环节进行了技术创新，为土壤中高溴代PBDEs的生物修复提供重要的参考依据。