Cheng Minggen, Meng Qiang, Yang Youjian, Chu Cuiwei, Chen Qing, Li Yi, Cheng Dan, Hong Qing, Yan Xin, He Jian
Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, China.
Provincial Key Laboratory of Agrobiology, Institute of Agro-biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China.
Appl Environ Microbiol. 2017 Mar 17;83(7). doi: 10.1128/AEM.03241-16. Print 2017 Apr 1.
Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through -dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of and are responsible for the initial step of MEA degradation in DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the -position. Transcription of was enhanced remarkably upon induction of MEA or DEA in DE-13. Additionally, and were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads. Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the -hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative.
在过去60年里，由于氯代乙酰苯胺类除草剂的广泛使用，细菌已经进化出了将这些化合物矿化的分解代谢途径。在上游分解代谢途径中，氯代乙酰苯胺类除草剂通过脱烷基化和酰胺水解转化为两种常见代谢物2-甲基-6-乙基苯胺（MEA）和2,6-二乙基苯胺（DEA）。MEA下游的途径由芳香环的羟基化启动，随后经过几步转化为用于环裂解的底物。该途径中的大多数关键基因已被鉴定。然而，参与MEA初始羟基化步骤的基因仍然未知。作为一种特殊的苯胺衍生物，MEA不能被已鉴定的苯胺双加氧酶转化。DE-13能够完全降解MEA并将其用作唯一碳源进行生长。在这项工作中，分离出了DE-13的一个MEA降解缺陷突变体。通过比较基因组分析预测了MEA分解代谢基因。遗传互补和异源表达的结果表明， 和 的产物负责DE-13中MEA降解的初始步骤。MeaXY是一种双组分黄素蛋白单加氧酶系统，它以NADH和黄素单核苷酸（FMN）作为辅因子催化MEA和DEA的羟基化。核磁共振（NMR）分析证实，MeaXY在 位使MEA和DEA羟基化。在DE-13中，MEA或DEA诱导后， 的转录显著增强。此外， 和 在其他降解MEA的鞘氨醇单胞菌中高度保守。这项研究填补了我们在鞘氨醇单胞菌中进行氯代乙酰苯胺类除草剂矿化的生化途径知识方面的空白。在过去60年里，人们一直非常关注氯代乙酰苯胺类除草剂的环境归宿。微生物降解被认为是这些化合物降解的重要机制。最近许多研究对氯代乙酰苯胺类除草剂的细菌降解进行了研究。已经获得了能够将这些除草剂矿化的纯培养物或混合培养物。已经提出了分解代谢途径，并且已经鉴定了大多数涉及的关键基因。然而，下游途径起始步骤（从MEA到羟基化MEA或从DEA到羟基化DEA）的负责基因尚未见报道。本研究表明，一种双组分黄素依赖性单加氧酶系统MeaXY催化鞘氨醇单胞菌中MEA或DEA的 羟基化。因此，这项工作找到了鞘氨醇单胞菌中进行氯代乙酰苯胺类除草剂矿化的生化途径中缺失的环节。此外，这些结果扩展了我们对一种特殊苯胺衍生物降解的理解。
