Study Of The Function Of MetF1 In The Tetrahydrofolate(THF)-Dependent Dicamba Demethylation System In Strain Ndbn-20

Dicamba(3,6-dichloro-2-methoxybenzoic acid)is a benzoic acid hormone herbicide widely used to control annual and perennial grasses in wheat,corn,sorghum and rice.Prevention and control of broadleaf weeds.With the emergence of more and more glyphosate-resistant weeds,and dicamba is characterized by low toxicity,high efficiency,and slow resistance,Therefore,dicamba is considered to be an ideal alternative to glyphosate herbicide-resistant transgenic target herbicides.In recent years,with the gradual popularization and use of gypsophila-resistant genetically modified crops,the use of dicamba in farmland environment will increase dramatically,bringing different degrees of damage to soil and water environment.Therefore,it is necessary to carry out the Study on the degradation and safety of dicamba in the environment.Studies have shown that microbial metabolism plays a crucial role in the degradation of dicamba.The tetrahydrofolate-dependent demethylase system is an important demethylation mechanism that is responsible for demethylation of dicamba.However,the properties and functions of the enzymes MetF1,FolD1 and PurU1 involved in the mechanism of tetrahydrofolate metabolism have not been elucidated.In this study,the dicamba degradation strain Rhizorhabdus dicambivorans Ndbn-20 was used as the research material to reveal the regeneration mechanism of tetrahydrofolate from the dicamba demethylation system in strain Ndbn-20 from the molecular biology level.The results of this paper are as follows:1、Study of the expression and function of MetFlPrevious studies showed that there are three THF-dependent methyltransferase gene clusters(scaffold 02,scaffold 50 and scaffold 66)in Ndbn-20 genome.In addition,bioinformatic analysis showed that there were five other putative THF metabolic genes(one metF,one folD and three purU)scattered in the genome.Under the condition of dicamba induction,only the scaffold 66 gene cluster was transcribed,and then the amino acid sequence phylogenetic tree analysis of the metF1-encoded protein located on the gene cluster revealed that MetF1 had the highest homology(66%)with 5,10-CH2-THF reductase MetF,whereas the similarity between MetFl and the reported E.coli MetF reductase is only 26%.And MetF from M chloromethanicum CM4 and S.paucimobilis SYK-6 are on a branch that participates in one-carbon metabolism of THF and catalyzes 5-CH3-THF to 5,10-CH2-THF,Therefore,it is preliminarily presumed that the physiological function of MetFl is 5-CH3-THF dehydrogenase activity.Exogenous expression of MetFl and E.coli MetF using the pET29a(+)expression system that reveals the quaternary structure of purified MetF1 is a monomer that binds to FAD coenzyme and plays 5-CH3-THF dehydrogenase activity in vitro and product was identified as THF and formaldehyde(5,10-CH2-THF is unstable,spontaneous decomposition).The Kcat and Km for 5-CH3-THF were 0.23±0.01 s-1 and 16.48±1.81 μM,respectively.However,The activity of 5,10-CH2-THF reductase was not detected.2、Preliminary identification of the function of metF1 in strain Ndbn-20 cellsThe genes both metFl and folDl in the THF-dependent demethylase gene cluster scaffold 66 were individually disrupted by homologous recombination single exchange technique.Two mutants,namely,Ndbn-20ΔmetF1 and Ndbn-20ΔfolD1 were generated.The enzymatic study found that the cell lysates of the mutant Ndbn-20ΔmetF1 completely lost the ability to 5-CH3-THF dehydrogenase activity,whereas the cell lysates of the mutant Ndbn-20ΔfolD1 and wild-type were completely transformed 5-CH3-THF to THF.It was further shown that MetF1 was the only active 5-CH3-THF dehydrogenase in cells grown on dicamba.3、Phenotypic analysis of metF1-disrupted mutant and verification of the function of dicamba-degradationThe cell lysates of the mutants Ndbn-20ΔfolD1 and wild-type Ndbn-20 can transform about 80%of dicamba,there was no significant difference in the rate of transformation,indicating that regeneration of THF does not require the involvement of the gene folD1.However,the cell lysates of Ndbn-20ΔmetF1 can only transform dicamba about 5%,the above results indicate that metFl is necessary for dicamba degradation,whereas folD1 is unnecessary.The colony morphology and growth rates of the mutants Ndbn-20ΔmetF1 and Ndbn-20ΔfolD1 were not different from wild-type,but whole-cell catalysis experiments showed that mutant Ndbn-20ΔmetF1 could not degrade dicamba.Both Ndbn-20ΔfolD1 and wild-type can be grown using dicamba as a carbon or source.Further explanation that the gene metF1 is necessary in the dicamba metabolic system,and the gene folD1 is unnecessary.Heterologous expression of the demethylase Dmt and 5-CH3-THF dehydrogenase MetF1,only when THF or 5-CH3-THF was present,simultaneously adding Dmt and MetF1 can quickly degrade dicamba.Above results indicate that THF can be regenerated by MetF1 catalyzed 5-CH3-THF produced by demethylation of dicamba in the dicamba demethylation system in vitro.The above studies revealed that the physiological function of MetF1 in the THF regeneration pathway of the R.dicambivorans Ndbn-20 strain dicamba demethylation system is 5-CH3-THF dehydrogenase irreversible catalysis of 5-CH3-THF conversion to 5,10-CH2-THF.