Cloning of a novel tetrahydrofolate-dependent dicamba demethylase gene from dicamba-degrading consortium and characterization of the gene product

Abstract

Dicamba, an important hormone-type systemic herbicide, is widely used to control more than 200 kinds of broadleaf weeds in agriculture. Due to its broad-spectrum, high efficiency and effectively killing glyphosate-resistant weeds, dicamba is considered as an excellent target herbicide for the engineering of herbicide-resistant crops. In this study, an efficient dicamba-degrading microbial consortium was enriched from soil collected from the outfall of a pesticide factory. The enriched consortium could almost completely degrade 500 mg/L of dicamba within 12 h of incubation. A novel tetrahydrofolate (THF)-dependent dicamba demethylase gene, named dmt06, was cloned from the total DNA of the enriched consortium. Dmt06 shared the highest identity (72.3%) with dicamba demethylase Dmt50 from Rhizorhabdus dicambivorans Ndbn-20. Dmt06 was expressed in Escherichia coli BL21 and purified to homogeneity using Co2+-charged nitrilotriacetic acid affinity chromatography. The purified Dmt06 catalyzed the transfer of methyl from dicamba to THF, generating the herbicidally inactive metabolite 3,6-dichlorosalicylate (3,6-DCSA) and 5-methyl-THF. The optimum pH and temperature for Dmt06 were detected to be 7.4 and 35°C, respectively. Under the optimal condition, the specific activity of Dmt06 reached 165 nmol/min/mg toward dicamba, which was much higher than that of Dmt and Dmt50. In conclusion, this study cloned a novel gene, dmt06, encoding an efficient THF-dependent dicamba demethylase, which was a good candidate for dicamba-resistant transgenic engineering.

Microbiology and Biochemistry of Pesticides Biodegradation

“…Genes involved in the biodegradation process of the chlorophenoxy herbicide Dicamba are situated in two different operons. In the first one, dmt genes encoding a demethylase are responsible for the first step in the biodegradation route of dicamba [372]; in the second operon, we found the genes dsmABC, dtdA, dsmD, dsmG, and dsmE that are responsible for the steps of reduction, oxidation, and dichlorination of the demethylated metabolite of dicamba. 2,4-D is another chlorophenoxy herbicide.…”