嗜热厌氧芽孢杆菌的偶氮染料降解分子机制及代谢途径解析

Azo dyes own multiple types and broad industrial application, however, huge amounts of hazardous wastewater are always very easily generated. Due to the lack of effective disposal measures, azo dye wastewater has caused serious environmental pollution problems. In the present study, a previously isolated thermophilic strain (Anoxybacillus sp. PDR2) with highly efficient azo dye degradation ability is used as the research target, where the whole genomic sequencing technique will be used to explore the functional genes related with the metabolism and environmental adaptation of the strain. On this basis, the key functional genes of this strain involved in the efficient degradation of azo dyes are identified through transcriptome sequencing and fluorescent quantitative real-time polymerase chain reaction (qRT-PCR) techniques, and the further cooperation with the investigation of the metabolic activities and azo dye degradative characteristics. Subsequently, physiological functions of the target genes during azo dye degradation process are explored through the knockout and complementation of the selected specific functional genes and the synchronous investigation of azo dye degradative characteristics. In addition, a possible metabolic pathway of azo dye Direct Black 19 was speculated by monitoring the intermediate metabolites produced by the strain PDR2 and its mutants. Finally, multic-omics techniques (including genomic, transcriptomic and metabolomics) and functional gene investigation are combined to clarify the molecular mechanism of azo dye degradation and metabolic pathways of the strain PDR2, which will further provide a theoretical basis for the highly efficient biotreatment of azo dye wastewater.

偶氮染料种类繁多应用广泛，但却极易产生巨量高毒废水，因缺乏有效的处置措施，从而对环境造成了严重的污染。本项目以前期分离得到的一株嗜热偶氮染料高效降解菌――厌氧芽孢杆菌PDR2为研究对象，通过全基因组测序发掘菌株与代谢及环境适应相关的功能基因，在此基础上进一步通过转录组测序以及实时荧光定量PCR技术并结合菌株的代谢活性和染料降解特性研究，确定菌株实现偶氮染料高效降解的关键功能基因。随后，通过对特定功能基因的敲除、回补及降解性能研究，探究目标基因在偶氮染料降解过程中的生理功能。此外，通过对菌株PDR2及其突变株降解偶氮染料直接黑19中间代谢产物的检测分析，推测菌株降解直接黑19的代谢途径；本研究最终通过基因组、转录组和代谢组等多组学技术以及功能基因研究等手段联合阐明菌株PDR2降解偶氮染料的分子机制和代谢途径，进而为偶氮染料废水的高效生物处置提供一定的理论研究依据。

偶氮染料种类繁多应用广泛，但却极易产生巨量高毒废水，因缺乏有效的处置措施，从而对环境造成了严重的污染。本项目以前期分离得到的一株嗜热偶氮染料高效降解菌――厌氧芽孢杆菌PDR2为研究对象，探究了菌株PDR2降解偶氮染料直接黑的最适条件及脱色特性。通过对菌株PDR2进行全基因组测序，发现菌株PDR2的基因组中含有多个编码趋化运动、热/冷休克蛋白、双组分系统、sigma因子、转座酶及整合酶等功能的基因，这些基因能够协助菌株PDR2适应偶氮染料的复杂环境。此外，对菌株PDR2产偶氮还原酶的产酶条件进行了优化，发现经优化后菌株酶活提升了43.16%，酶活高达634.20 U，随后克隆了菌株的两种偶氮还原酶并成功外源表达。然后，对偶氮还原酶进行同源建模与分子对接，发现上述偶氮还原酶对单偶氮键底物表现出了明显的偏好。此外，通过转录组比较偶氮染料胁迫下和正常葡萄糖培养下菌株PDR2的基因差异表达情况，再结合RT-qPCR技术发现编码NAD(P)H-黄素还原酶，2Fe-2S铁氧还蛋白和NAD(P)-依赖性脱氢酶的基因在偶氮染料降解过程中起重要作用。随后，通过多种现代生物学检测手段分析了DBG的降解产物、推测了其降解途径，且验证了DBG被菌株PDR2降解后脱毒的效果；通过非靶向代谢组学和蛋白质组学技术对不同降解时期偶氮染料胁迫和正常葡萄糖培养下菌株PDR2体内代谢物和蛋白进行进行差异分析，发现在偶氮染料降解的过程中有机酸及其衍生物、核苷酸脱氧核苷酸及其衍生物等类物质为主要差异代谢产物，且菌株PDR2对偶氮染料的降解过程主要取决于细胞质中蛋白质的变化，且大多数蛋白存在于与碳水化合物代谢有关的通路中；最后，通过联合分析发现菌株先通过分泌核黄素作为氧化还原介体加速电子供体的电子向电子受体传递，并通过自身生长呼吸的代谢通路为偶氮染料还原降解所需的电子提供了来源，之后菌体大量分泌氨基酸分子减少偶氮染料及其降解产物对菌体的毒害作用，同时又为合成相关蛋白提供原料，加快合成效率，使偶氮染料的降解更加高效彻底。上述研究结果可为偶氮染料废水的高效生物处置提供一定的理论研究依据。