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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.

SUBMITTER: Li N

PROVIDER: S-EPMC9404685 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Li Na N Chen Le L Chen E E Yuan Cansheng C Zhang Hao H He Jian J

Frontiers in microbiology 20220811

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 ...[more]

PMID: 36033860

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Project description:UnlabelledSphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops.ImportanceDicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.

Project description:The herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system in Rhizorhabdus dicambivorans Ndbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely, metF (coding for 5,10-CH2-THF reductase), folD (coding for 5,10-CH2-THF dehydrogenase-5,10-methenyl-THF cyclohydrolase), and purU (coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. The metF gene of scaffold 66 (metF1) was expressed in Escherichia coli BL21(DE3), and the product was purified as a His6-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH3-THF dehydrogenase activity in vitro The kcat and Km for 5-CH3-THF were 0.23 s-1 and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed that metF1 is essential for dicamba degradation, whereas folD1 is dispensable.IMPORTANCE There are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome of R. dicambivorans Ndbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF-dependent dicamba demethylation system in R. dicambivorans Ndbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1 in vitro did not show 5,10-CH2-THF reductase activity, which is the physiological function of Escherichia coli MetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.

Dataset Information

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

Publications

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

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