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

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.

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。

广泛施用的拟除虫菊酯类农药（PYRs）降解的必经中间产物—3-苯氧基苯甲酸（3-PBA）在造成农产品二次污染的同时，也会导致其母体农药生物矿化降解受阻，间接地使农药残留问题更加严峻。本项目重点研究了食品源高效降解3-PBA菌株的特性、途径及机理。米曲霉M4在3-PBA降解初期可将其短暂还原生成毒性相对较低的3-苯氧基苄醇（3-PBlc），随3-PBA羟基化生成水溶性更强的HO-3-PBA，3-PBlc又转化生成3-PBA，最终实现3-PBA完全降解，下游产物HO-3-PBA进一步羟基化生成(HO)2-3-PBA。3-PBA降解率不完全与M4菌体生物量成正比，富集培养基较营养更充分的马铃薯液体培养基更利于M4对3-PBA的降解。0.1 g/L Fe2(SO4)3、MnSO4、ZnSO4可分别促进M4的生长及其对3-PBA的降解，三种金属离子复合使用促进作用更加显著。初始高剂量3-PBA（约100 mg/L）较低剂量（约25 mg/L）可诱导M4生长态细胞和静息细胞代谢合成更多参与3-PBA还原和羟基化的酶。通过全基因组及转录组生物信息学分析，结果表明M4生长态细胞从氧化应激、DNA修复和膜转运三个方面参与了3-PBA应激解毒。M4静息细胞从氧化应激和膜转运两方面参与3-PBA应激解毒，3-PBA对静息细胞基因毒性作用较生长态细胞小。同时，从鞘氨醇单胞菌SC-1中筛选到降解3-PBA关键酶基因sca，其基因工程菌全细胞、静息细胞、全细胞破碎液均可降解3-PBA，将细胞破碎液离心分离得到细胞破碎上清和细胞碎片液对3-PBA的降解作用不理想。静息细胞可降解4'-HO-3-PBA和3'-HO-4-PBA，但不降解氟磺胺草醚，推测该基因具有断裂与3-PBA结构类似物的二苯醚键的功能。项目研究成果为食品和饲料中残留PYRs和3-PBA的减除提供了重要的理论基础和食品用安全微生物资源。