土霉素高效降解菌ZAF-05的降解途径及分子机制研究

As one of the most used tetracycline antibiotics, oxytetracycline now is widespread used in poultry and livestock breeding in China and its residuals are of great harm. In natural environment, microbial degradation is the main denature pathway of oxytetracycline, however rare researches are conducted about its metabolic mechanism until now. The applicant of this project has obtained one highly efficient degrading strain ZAF-05 that can utilize oxytetracycline as the sole carbon source and energy. Based on this, we focus on sequencing and analyzing the genome of the strain, as well as comparing the different transcriptomics when the strain cultured with oxytetracycline and glucose as the solo carbon source respectively, aiming to excavate degradation-related genes, verify the function of key genes using molecular biology technology and finally build gene expression network model according to bioinformatic analysis. Moreover, with the analysis of metabolism process by HPLC-MS/MS, the complete degradation pathway and molecular regulation mechanisms of oxytetracycline degrading in strain ZAF-05 will be demonstrated. The study will enrich mechanism research in the field of oxytetracycline degradation by microorganisms and provides theoretical basis and technical guidance for the biological control and bioremediation of environmental oxytetracycline.

土霉素作为四环素族抗生素的主要类型在我国禽畜养殖业大范围使用，残留危害严重。自然环境中的微生物降解是去除残留的主要手段，但迄今为止其代谢机制却鲜有研究。本项目申请者在筛选获得能以土霉素为唯一碳源和能源生长的菌株ZAF-05的基础上，拟通过对菌株进行基因组测序与基因功能的分析、注释以及对不同底物（葡萄糖和土霉素）条件下其差异转录组的分析比较，挖掘参与降解的关键基因，经分子生物学技术对所得基因进行功能验证，根据生物信息学技术构建土霉素降解的基因调控网络；同时结合HPLC-MS/MS手段对菌株降解过程的全面解析，摸清菌株对土霉素的降解途径及分子调控机制。实验结果对于丰富微生物降解土霉素的机理研究具有重要的意义，同时也为环境中土霉素残留的生物控制和生物修复提供理论基础和技术指导。

由于禽畜养殖业的大范围使用，我国土霉素残留危害严重。利用微生物降解是去除残留的有效手段，但迄今为止其代谢机制和分子机理却鲜有报道。本项目以从制药厂污泥中分离获得的能高效降解土霉素的菌株ZAF-05为研究对象，通过对降解特性的研究明确了菌株发挥降解作用的初始土霉素浓度、温度以及pH 等环境适应性；利用传统结合分子手段明确了菌株为Arthrobacter nicotianae；通定期检测土霉素在降解体系中的代谢产物“碎片”，推导出土霉素经加氧、脱甲基、脱羧降低毒性；对菌株进行基因组测序与基因功能的分析，发现该菌株有23个基因与抗生素抗性相关，此外还包括与降解相关的加氧酶基因和与解毒作用相关的体外排泵和β内酰胺酶基因。实验结果对于丰富微生物降解土霉素的机理研究具有重要的意义，同时也为环境中土霉素残留的生物控制和生物修复提供理论基础和技术指导。