title: Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA.
abstract: 2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02' in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Qiongzhen
authors.initials: Q
authors.lastname: Chen
authors.affiliation: College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Shuhuan
authors.initials: S
authors.lastname: Li
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhuang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Huang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Zhoukun
authors.initials: Z
authors.lastname: Li
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Jue
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Fu
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Zhengguang
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000081:Acetamides / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D004269:DNA, Bacterial / Q000235:genetics; D042742:Delftia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010449:Peptide Mapping; D016958:Pseudomonas putida / Q000378:metabolism; D017422:Sequence Analysis, DNA; D042301:Sphingomonadaceae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014052:Toluidines / Q000378:metabolism*
chemical_list: D000081:Acetamides; D004269:DNA, Bacterial; D014052:Toluidines; C000610366:2-methyl-6-ethylaniline; C078188:2-chloro-N-(ethoxymethyl)-N-(2-methyl-6-(trifluoromethyl)phenyl)acetamide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)