辅因子介导人工混菌降解磺胺甲恶唑研究

The widespread use and disposal of sulfonamide antibiotics has led to serious environmental pollution in human medicine and livestock and poultry breeding. The biodegradation of sulfonamide antibiotics by natural microbial consortia is through multiple metabolic pathways, which severely limit the efficiency and directed regulation of the biodegradability of some antibiotics. The stability and function of the microbial community depends on the material interactions.. In this work, to understand the low efficiency and poor controllability of sulfonamide antibiotics degradation, we will focus on the key issue of the regulatory roles of cofactors in the biodegradation of sulfamethoxazole (SMX) with Engineering Microbial Consortia (EMC). Firstly, the kinds and levels of cofactors are mediated by reconstituting or building functional modules in EMC. The relationship between cofactor cross-feeding and SMX degradation was explored by measuring the changes in the levels of cofactors and SMX. Furthermore, we investigate the changes in energy metabolism and redox status, and explore the changes in metabolome, stability, and SMX biodegradability of EMC, so as to reveal the potential relationship among the fitness and stability of EMC, cell-to-cell communication and SMX biodegradation. Additionally, the potential metabolic pathway of SMX is also explored through detecting the intermediate metabolites and the final degraded products during SMX biodegradation. Taken together, these results will elucidate the mechanism of material exchange in EMC when cofactors efficiently regulate the SMX biodegradation. Moreover, through evaluating the biodegradability of SMX in EMC, it will provide a theoretical basis for reconstituting EMC and microbial agents to efficiently degrade SMX and the strategies for eliminating the sulfonamide antibiotics in environments.

在医疗和畜禽养殖业中磺胺类抗生素大量使用造成了严重的环境污染。天然混合菌中抗生素降解的多代谢途径难以定向调控，限制了微生物对其的高效降解。微生物群落的稳定性和功能取决于菌群落间物质相互作用。. 本项目针对天然混合菌群降解磺胺类抗生素效率低和可控性差等问题，提出构建辅因子介导人工混菌降解磺胺甲恶唑(SMX)的科学设想，从不同微生物间物质交流入手，分析物质交流与菌群稳定性间关系；寻找辅因子变化规律，运用合成生物学方法构建辅因子功能模块，调节菌群间辅因子种类和浓度，揭示辅因子饲喂与SMX降解速率间潜在关系；检测SMX的中间代谢物，寻找潜在的可控代谢途径；探究细胞代谢物与菌群稳定性和SMX降解速率变化规律，以揭示菌群适配性、物质交流与SMX降解间关系，以期阐明辅因子调控SMX生物降解的物质交流机制。研究结果为构建高效人工混菌降解SMX和菌剂开发提供理论依据，为解除环境中磺胺类抗生素寻找新策略。

磺胺类抗生素在医疗和畜禽养殖业中大量使用造成了严重的环境污染, 导致抗生素耐药性菌株，严重威胁到人类健康。天然或人工驯化的微生物混合菌群存在运行周期长、转化效率低、稳定性及可控性差等问题，本项目通过构建人工混菌体系高效移除磺胺甲恶唑（SMX）等抗生素。研究结果表明：.粪产碱杆菌（AF菌）利用SMX作为碳源，混菌移除低浓度SMX（10 mg/L和50 mg/L）效率达90%，SMX提高了混菌与AF菌胞内NADH/NAD+的水平，乙酰化SMX是混菌在摇瓶培养条件下的主要转化物；中空纤维膜生物反应系统有利于SMX的去除，降低乙酰化和羟胺化SMX的形成。.高浓度的外源VC、VB6和GSSG提高了AF菌SMX的去除率，高浓度的VC、NADH和NAD+促进HO-SMX的形成，VB12抑制HO-SMX的形成，低含量GSSG促进Ac-SMX的形成，SMX的去移除可能与AF菌的生物量活性。.AF菌和血红密孔菌混菌体系减轻SMX转化产物的细胞毒性，提高了SMX的去除效率，抑制了HO-SMX的形成。在此系统中SMX主要通过生物转化而不是吸收。血红密孔菌在两天内完全清除SMX、环丙沙星（CIP）和诺氟沙星（NOR）的混合物, 血红密孔菌和黄孢原毛平革菌纯培养和共培养消除SMX、CIP和NOR的抗菌活性，共培养是移除SMX和NOR的一种更环保的策略。.同时，发现益生菌克劳氏芽孢杆菌在8小时内除去头孢呋辛、头孢噻肟和头孢吡肟100%的混合物，头孢呋肟和头孢吡肟在共培养条件下的生物毒性低于纯培养。生物表面活性剂对TCs的去除效果优于非离子表面活性剂, 四环素类抗生素的生物降解效率依次为氯四环素（CTC）>氧四环素（OTC）>四环素（TC）, TCs的去除主要是通过生物转化，而不是吸附和水解, 共培养减轻OTC和CTC转化产物的细胞毒性。 .这些研究为环境中SMX等抗生素降解提供了重要的理论依据，为构建人工混菌系统高效生物移除SMX等抗生素提供了新思路。