细菌代谢吡虫啉的羟基化和硝基还原途径中的关键酶和调节机制研究

The worldwide used neionicotinoid insecticide imidacloprid has caused serious environmental and ecological problems. The research group found that Stenotrophomonas maltophilia CGMCC 1.1788，Pseudomonas putida KT2440 and Escherichia coli DH10B co-metabolized imidacloprid via nitroreduction and/or hydroxylation pathways. However the key enzymes of these two pathways have not been elucidated. The proposers also found that the hydroxylation of imidacloprid by S. maltophilia CGMCC 1.1788 was regulated by the primary energy substrates carbohydrate and organic acid. The mechanism was that the enzyme activity of 5-hydroxy IMI dehydratase was inhibited by cofactor NADPH regenerated from carbohydrate metabolism, while not affected by NADH and even enhanced by organic acid itself, which therefore affected the production of olefin imidacloprid, a metabolite with 19-fold higher insecticidal activity than imidacloprid. In this proposal, the above three imidacloprid-degrading bacteria will be used as research objects to identify the imidacloprid nitroreductase and hydroxylase by the methods of gene knockout and retro-complementation as well as gene cloning and overexpression. The 5-hydroxy imidacloprid dehydratase is identified by enzyme purification and mass spectrum analysis and then this identified enzyme is overexpressed and purified, and examined its enzymatic characters. The regulation manner of 5-hydroxy imidacloprid dehydratase by NADPH and organic acid is analyzed by molecular docking and bioinformatic analysis. The NADPH binding domain of 5-hydroxy imidacloprid dehydratase is mutated to explore whether the NADPH inhibitory effect of the mutated enzyme is terminated. These works are very important to understand the mechanism of microbial co-metabolic degradation of organic contaminants and decrease the imidacloprid residue in environments.

烟碱类杀虫剂吡虫啉的广泛施用已引起了严重的环境生态问题。课题组发现嗜麦芽寡养单胞菌CGMCC 1.1788、恶臭假单胞菌KT2440和大肠杆菌DH10B可共代谢降解吡虫啉，代谢途径为羟基化或/和硝基还原，但关键代谢酶尚不清楚；嗜麦芽寡养单胞菌的吡虫啉羟基化途径受糖和有机酸调节，其机制是糖代谢产生的NADPH抑制5-羟基吡虫啉脱水酶活性，NADH不抑制，有机酸反而促进该酶活性，从而影响了杀虫活性比吡虫啉高19倍的烯式吡虫啉的生成。本课题以上述三个菌株为研究对象，进行基因敲除与回补和克隆与表达，鉴定吡虫啉硝基还原酶和羟基化酶；通过酶分离纯化和质谱分析鉴定5-羟基吡虫啉脱水酶，表达和纯化该酶并分析酶学特性，采用分子对接和生物信息学分析NADPH和有机酸调节酶活性的方式，对NADPH结合结构域点突变，分析突变酶能否解除NADPH抑制。本研究对认识微生物共代谢降解现象和降低吡虫啉的残留有重要意义。

烟碱类杀虫剂吡虫啉(imidacloprid)的广泛施用产生了较严重的环境生态问题。微生物降解法是消除其环境污染的主要方法之一，开展微生物降解吡虫啉的研究有助于消除其环境污染和降低其生态风险。课题组发现假黄单胞菌Pseudoxanthomonas indica CGMCC 6648、恶臭假单胞菌Pseudomonas putida KT2440、嗜麦芽寡养单胞菌Stenotrophomonas maltophilia CGMCC 1.1788和大肠杆菌Escherichia coli DH10B可经由羟基化或/和硝基还原途径降解吡虫啉，本课题采用基因克隆与表达、蛋白分离纯化、基因组学和蛋白质组学，HPLC分析等方法开展了吡虫啉羟基化酶、5-羟基吡虫啉脱水酶和吡虫啉硝基还原酶的鉴定和调控机制研究。课题组发现不同培养时间的菌体的羟基化活性不同， 培养18 h的P. indica CGMCC 6648的吡虫啉羟基化活性是培养4h的菌体的4倍，而S. maltophilia CGMCC 1.1788的吡虫啉羟化活性则不受细胞培养时间的影响。基因克隆和表达表明P. putida KT2440的乙醛氧化酶为吡虫啉硝基还原酶。从水体中筛选获得一株具有较高吡虫啉降解能力的寡营养细菌薄层菌H. latericoloratus CGMCC 16346。以麦芽糖为共代谢基质，该菌株的静息细胞在6d内可降解64.4%的100 mg/L吡虫啉；其生长细胞在10d内可降解40.8%吡虫啉。基因组测序和COG分析表明，CGMCC 16346是寡营养细菌，适应了低营养的水体环境生长。H. latericoloratus CGMCC 16346在水体中降解吡虫啉不需要添加共代谢基质即可降解吡虫啉，且接种30d后仍具有较好的吡虫啉降解能力。这一特性使其具有用于水体修复吡虫啉的应用价值。课题组还筛选到一株高效降解另一种广泛施用的烟碱类杀虫剂啶虫脒(acetamiprid)的放线菌暗灰链霉菌Streptomyces canus CGMCC 13662。该菌株的降解酶为腈水合酶，该腈水合酶由三亚基组成，且没有腈水合酶成熟所需的激活蛋白。