微生物降解恩诺沙星的分子机理研究

Enrofloxacin, one kind of the fluoroquinolone antibiotics, has been extensively applied in the intensive aquaculture industry. Its residue has been frequently detected in food samples, the environment and even human body, endangering the ecosystem and human health. The application of microorganism-based enzymatic treatment to remove enrofloxacin residues in the environment has been considered an environmentally friendly, efficient as well as safe way. Toward this end, elucidating the enrofloxacin-degradative pathway and its molecular mechanism is greatly needed. However, the molecular mechanism of microbial degradation of enrofloxacin has not yet been reported. Based on the fact we have obtained enrofloxacin-degrading consortium and excuted preliminary exploration of enrofloxacin degradation pathway, the aim of this study was to either isolate enrofloxacin-degrading pure culture and to identify the catabolic genes from the pure culture through genome/transcriptome sequencing as well as construction of transposon-mediated mutation library or clone the catabolic genes from the pre-existing consortium via omic techniques. Subsequently, the function of key catabolic gene and/or corresponding gene product was characterized via gene knock-out/complementation, mass-spectrometric technique and so on to elucidate the catabolic pathway at molecular and enzymatic levels. The results obtained in this work would advance progress in elucidating enrofloxacin degradation pathway and, most importantly, fill a gap in the understanding of molecular mechanism behind enrofloxacin catabolism, by which lay a foundation for the use of enzymatic treatment to eliminate enrofloxacin residues in the environment.

恩诺沙星（氟喹诺酮类抗生素）被广泛应用到集约化养殖产业；其残留常检测于食品、环境样品及人体内，危害生态平衡和人体健康。应用微生物酶处理技术去除恩诺沙星残留是一种环境友好型且高效安全的修复方法。解析恩诺沙星微生物降解途径及其分子机理是将该方法付诸实践的前提。然而，目前尚无微生物降解恩诺沙星分子机理的研究报道。因此，在已获得具有恩诺沙星降解能力的混合菌群及初步探究了其对恩诺沙星代谢途径的工作基础上，本项目拟从中筛选降解菌株并通过基因组/转录组测序及构建转座子插入突变文库等方法从降解纯菌或借助组学技术从上述混合菌群中克隆恩诺沙星关键降解酶编码基因；后续借助基因敲除及回补、质谱分析等方法对关键基因及其编码产物进行功能鉴定，以期阐明恩诺沙星的代谢途径及其分子机理。本研究结果将完善恩诺沙星降解途径并填补对其代谢分子机理的认知空白，继而为应用微生物酶处理技术修复恩诺沙星残留奠定理论基础。

兽用抗生素在集约化的水产养殖业中作用显著，能够预防、治疗动物疾病并促进动物生长。由于养殖对象对抗生素的吸收率有限，兽用抗生素残留常被检测于食品、环境样品甚至人体内。喹诺酮类抗生素（尤其是恩诺沙星）是上海地区养殖环境中检出率较高的抗生素，危害生态平衡和人体健康。团队前期的研究证明了种植沉水植物苦草生态修复养殖池塘污染底泥的可行性，然而修复机理尚未明确。在自然环境中，微生物在降解或转化有毒化合物中起到举足轻重的作用。鉴于此，本项目收集生态修复实验结束后的苦草根际沉积物，从中分离到一株新的恩诺沙星降解真菌Humicola sp. KC0924g，研究了其降解特性。菌株KC0924g除了可降解喹诺酮类抗生素（恩诺沙星、环丙沙星、诺氟沙星和氧氟沙星）外，还可降解水产养殖禁用药（孔雀石绿和隐性孔雀石绿）。根据UHPLC-MS/MS鉴定结果，推测了菌株KC0924g分别通过4条途径降解恩诺沙星，即乙基哌嗪环的开裂途径（pathway I）、苯环的羟基化途径（pathway II）、氧化脱羧途径（pathway III）和脱氟途径（pathway IV）。其中，在脱氟途径中（pathway IV），推测到一条新的降解支路。Cu2+（500 μM）的诱导可以显著性的提升菌株KC0924g对恩诺沙星的降解效率并缩短降解半衰期。漆酶抑制剂和细胞色素P450抑制剂均在一定程度上抑制了菌株降解恩诺沙星的效率。结合比较转录组分析（比较非诱导组和Cu2+诱导组）结果，我们发现一个FAD-依赖型单加氧酶编码基因（DN2180），一个漆酶编码基因（DN6638）和两个细胞色素P450酶编码基因（DN16444和DN21088）与恩诺沙星降解相关。总体来看，研究结果加深了我们对微生物降解恩诺沙星的认识，为水产养殖环境中喹诺酮类抗生素的修复应用提供了优良的菌株资源，为后续研究微生物-植物根际互作提供了实验材料。