米曲霉M-4降解3-苯氧基苯甲酸关键酶的基因筛选及其酶解机理研究

拟除虫菊酯类农药是目前广泛施用的杀虫剂，该类农药降解的必经中间产物—3-苯氧基苯甲酸（3-PBA）结构非常稳定，会导致其母体农药的生物矿化降解受阻和农产品二次污染，间接地使农药残留问题更加严峻。国内外目前尚未对3-PBA降解酶开展深入系统的研究。本项目拟以源于酱油曲的3-PBA高效降解菌—米曲霉M-4（Asp. oryzae M-4）为对象，利用现代分子生物学和生化相关技术，开展全基因组和转录组测序与基因注释，筛选3-PBA降解关键酶候选基因，采用酶基因克隆表达和基因敲除技术，确定3-PBA降解关键酶，研究酶结构与酶学特性，阐明发酵食品源米曲霉M-4对拟除虫菊酯类农药及其关键中间产物3-PBA的降解机理。该研究成果不仅可为食品中残留拟除虫菊酯类农药及3-PBA的生物降解与控制奠定理论基础，而且可为3-PBA结构类似物的其他二苯醚类农药的酶法降解提供借鉴，具有重要的理论意义和应用价值。

Pyrethroid pesticides have been widely applied in agricultural production. The major metabolite of pyrethroids, 3-phenoxybenzoic acid (3-PBA), is chemically stable and easily accumulates in various environments, which hinders the pathway by which its parent pesticides are completely degraded into non-toxic small molecules, thus indirectly making the problem about pesticide residues in food more serious. The enzymes and relevant encoding genes for degrading 3-PBA are largely unstudied. To address this gap, based on highly efficient 3-PBA-degrading strain Aspergillus oryzae M-4 isolated from soy sauce koji, we utilized a series of modern molecular biology and biochemistry techniques to carry out whole genome and whole transcriptome sequencing and gene annotations, to screen tentative genes encoding key enzymes for degrading 3-PBA, to clone and express 3-PBA-degrading enzymes, in combination with gene knockout technique for finally double-checking the key degrading enzymes, as well as biochemical tools for enzymatic structure and characteristics, ultimately illuminating the biodegradation mechanism of pyrethroids and 3-PBA by fermented foods-origin M-4. The results will not only lay a foundation for biodegradation and control of pyrethroid residues and 3-PBA in foodstuffs, but also provide guidance for the enzymatic degradation of other categories of diphenyl ether pesticides, the analogues of 3-PBA. Therefore, the research possesses both considerable theoretical and practical values.