Aeromonas sp. DH-6的染料广谱代谢分子机理及电子传递的作用

The toxicity and recalcitrant of azo- and triphenylmethane dyes increase the risk to human health and ecosystems. The dye degradation by most of bacterial Azoreductase and Triphenylmethane reductase is specific, which inhibit their practical application. In this project, the new type of broad-spectrum metabolism enzyme from Aeromonas sp. DH-6 is hoped to solve the problem. Therefore, this study will: 1) Construct the random mutation library by transposon mutagenesis method, and find out the functional genes relative to broad-spectrum metabolism via TAIL-PCR and chromosome walking techniques after comparison with the framework of whole-genome; 2) BioBrick was used to recombine potentially catalytic related genes into a series of different transcription units, and express these genes in E. coli to figure out the relationship of each gene coding enzymes in the catalytic pathway, and locate the key enzymes to the metabolic pathway; 3) explore the involvement of electron transfer in the process of broad-spectrum metabolism of dyes by strain DH-6 using the electronic mediator; 4) Detailedly analyze and identify the intermediates of Malachite Green and Methyl Orange degradation by strain DH-6, and propose its possible biochemical metabolic pathway; further to elucidated the molecular mechanism of efficient and broad-spectrum dye metabolism by bacteria. This research will show the molecular mechanism of bacterial dye metabolism with broad-spectrum, draw the accurate and detailed degradation pathway, and finally provide the scientific basis for the feasibility and applicable prospects for dye biodegradation.

偶氮和三苯甲烷类染料的毒性、难降解性给人类健康和生态系统带来了潜在的巨大风险。已知的细菌偶氮还原酶和三苯甲烷还原酶底物专一性强，限制了其实际应用，而本研究分离筛选到的高效广谱染料降解气单胞菌DH-6中的新型广谱代谢酶则有望解决该问题。因此，本项目拟：1）以转座子突变方法构建随机突变库，通过TAIL-PCR和染色体步移技术且经全基因组框架图比对后，鉴定广谱代谢相关功能基因；2）采用BioBrick将催化相关基因重组为一系列差异性转录单元，并表达于大肠杆菌，理清各基因编码蛋白在催化途径中的顺序关系；3）采用加入电子介体方式，探索电子传递在菌株DH-6广谱代谢染料过程中的作用；4）分析鉴定孔雀石绿和甲基橙的代谢中间产物，推测其生化代谢途径，进而详尽阐明细菌广谱降解染料的分子机制。本研究工作的开展将解析细菌高效广谱降解染料的代谢通路及分子机制，从而为染料生物降解技术的可行性、发展前景提供科学依据。

偶氮和三苯甲烷类染料的毒性、难降解性给人类健康和生态系统带来了潜在的巨大风险。已知的细菌偶氮还原酶和三苯甲烷还原酶底物专一性强，限制了其实际应用，而本研究分离筛选到的高效广谱染料降解气单胞菌DH-6中的新型广谱代谢酶则有望解决该问题，并将填补国际上细菌降解染料详细分子机制领域的空白。项目采用全基因组测序、转录组测序和实时荧光定量PCR技术相结合，探索菌株DH-6参与降解甲基橙染料和孔雀石绿染料的相关基因。研究结果表明，菌株DH-6的基因组由一条3006655bp的完整环状染色体组成，GC含量为56.68%。通过生物信息学分析，进一步阐明了菌株DH-6降解偶氮染料和三苯甲烷染料的遗传基础及其适应染料环境的能力。RNA-Seq和qRT-PCR技术证实，菌株DH-6表达的细胞色素P450、NAD(P)h-黄素还原酶、和NAD(P)依赖的乙醇脱氢酶基因是MG和MO降解的关键基因。通过加入电子介体蒽醌（AQDS）作为唯一电子受体，探明了电子传递在菌株DH-6广谱代谢染料过程中的促进作用。此外，采用UV/Vis、FT-IR、NMR、GC-MS和LC-MS技术相结合，分析了各催化酶对MO和MG的代谢中间产物，并通过HPLC分离纯化代谢中间产物后与标准化合物进行比对分析，确定了代谢中间产物，最终勾勒出Aeromonas sp. DH-6中的染料分子广谱代谢途径，为微生物降解染料的分子机制研究提供了理论依据。

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