background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown.
results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, ... More
background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown.
results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCD), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthR), a ferredoxin reductase (EthA), a cytochrome P450 monooxygenase (EthB), a ferredoxin (EthC) and a 10-kDa protein of unknown function (EthD). Complementation with EthABCD and EthABD, but not EthABC in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCD was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCD or EthABD but not EthABC catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABD displayed maximal activity at 30 °C and pH 7.5.
conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.
