基于转录组学和蛋白质组学解析Sphingopyxis sp. YMCD对微囊藻毒素的 代谢解毒机理

Microcystin (MC) poses a great threat to drinking water safety and public health. Biodegradation with the participation of bacteria is one of the dominant processes of MC removal and detoxification in the water environment. However, relatively few MC-degrading bacteria have been isolated and had their mechanisms reported, which limited the development of MC removal by environmental bacteria. A novel indigenous microcystin-degrading bacterium Sphingopyxis sp. YMCD was isolat- ed and its complete genome sequence was obtained for the first time by us (acces- sion number KY491642 in the Genbank). According to multi-interdisciplinary knowledge of Environmental Health, Bioinformatics, Analytical Chemistry, Microbi- ology, Toxicity, and Molecular Biology, the microcystin-detoxifying mechanisms of Sphingopyxis sp. YMCD will be investigated in this study. First of all, the microcystin-detoxifying pathway will be studied by identifying the degradation products using high performance liquid/gas chromatograpy coupled with mass spectrometry (HPLC/GC-MS).What’s more, the molecular-biological mechanisms of this bacterial strain against MC will be analyzed through combining transcript- omics and proteomics results, which are obtained using the high-throughput sequencing and Tandem Mass Tags (TMT) protein labeling methods. The microcystin- degrading genes and proteins will be found and their characteristics will be studied through expressing the microcystin-degrading substances in the genetically engineered bacterium. Last but not the least, the microcystin-detoxifying effects of this bacterial strain will be estimated by analyzing the individual and joint toxicities of the degradation products. In conclusion, some novel degradation products, microcystin-degrading proteins, and genes will be obtained in this study, which can enrich knowledge of microcystin-detoxifying mechanisms of micro- organism. Moreover, the study can provide a scientific basis and technical support for removing MC safely and efficiently by environmental bacteria.

微囊藻毒素（Microcystin，MC）严重威胁饮用水安全和人类健康，微生物代谢降解是安全高效去除MC的主要方法之一，但微生物降解MC的机理尚未明确，限制了生物防治MC技术的发展。我们在率先获得高效MC降解菌Sphingopyxis sp. YMCD及其全基因组序列的基础上，通过多学科交叉，开展三方面研究：①采用色谱-质谱联用等技术纯化和鉴定MC降解产物，阐明此菌代谢降解MC的途径；②采用高通量测序和定量蛋白质组学技术分别获得该菌降解MC不同阶段的转录组与蛋白质组数据，通过多组学联合分析，筛选出MC代谢降解蛋白及其编码基因，并利用蛋白体外表达技术验证其生物学功能，揭示此菌代谢降解MC的分子机制；③研究MC降解产物的毒性，探明此菌代谢解毒MC的效果。本课题将发现新的MC降解产物、代谢降解蛋白和基因，阐明YMCD菌对MC的代谢解毒机理，为构建安全高效的生物防治MC技术提供科学依据与技术支持。

目的：探讨Sphingopyxis sp. YF1（原名YMCD）代谢降解微囊藻毒素MC-LR的途径，阐明其代谢降解的分子生物学机制，探讨MC-LR及其降解产物的肝毒性作用及机制，评估该菌代谢解毒MC-LR的效果。材料与方法：采用色谱-质谱联用等技术纯化鉴定MC降解产物，阐明此菌代谢降解MC-LR的途径；采用高通量测序和蛋白质组学技术获得该菌降解MC-LR的组学数据，通过联合分析筛选MC-LR代谢降解蛋白及其编码基因，结合qPCR和蛋白体外表达技术验证其生物学功能，通过同源建模与分子对接技术揭示关键降解蛋白酶与底物的结合模式及结合位点；使用试剂盒检测PP2A活性，通过qPCR及WB等技术，验证miR-451a及SREBP1在MC-LR所致的肝损伤中的作用及机制。结果：YF1代谢降解MC-LR的最大速率可达5 mg/mL/h，YF1中MlrA、MlrB、MlrC酶可逐步将MC-LR降解为线性MC-LR、四肽和Adda。Adda进一步被降解为苯乙酸PAA后，PAA被PAAase转化为PAA-CoA，PAA-CoA被PaaA、PaaG和PaaZ同源酶降解为CoA，CoA通过三羧酸循环完全转化为CO2。这些酶通过氢键、范德华力等作用力与底物结合降解MC-LR，且pH、MC-LR浓度和MlrA酶浓度均可影响MlrA酶降解MC-LR的效率。与MC-LR单独处理组相比，MC-LR的中间降解产物明显缓解了MC-LR对PP2A活性的抑制。MC-LR处理导致miR-451a及SREBP1表达改变。结论：YF1可彻底降解MC-LR；明确了YF1菌代谢降解MC-LR的分子生物学机制；MC-LR可调控miR-451a及SREBP1影响MC-LR诱导的肝脏损伤；MC-LR的中间降解产物以及终产物CO2的肝细胞毒性远小于MC-LR，YF1可解毒MC-LR。该研究为构建安全高效的生物防治微囊藻毒素技术提供科学依据和技术支持，具有较强的应用前景。