ABSTRACT
The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.
Subject(s)
Carboxylesterase/genetics , Carboxylesterase/metabolism , Herbicides/metabolism , Oxazoles/metabolism , Propionates/metabolism , Rhodococcus/isolation & purification , Rhodococcus/metabolism , Biotransformation , Cloning, Molecular , Cluster Analysis , Carboxylesterase/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Molecular Sequence Data , Molecular Weight , Phylogeny , /genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/enzymology , Rhodococcus/genetics , Sequence Analysis, DNA , Soil Microbiology , Substrate Specificity , Triticum/growth & developmentABSTRACT
Acetohydroxyacid synthase (AHAS) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. AHAS is the target for sulfonylurea, imidazolinone and other AHAS-inhibitor herbicides. Herbicides-resistant AHAS genes have potential application in plant transgenetic engineering and development of new generation herbicide. The AHAS isozyme genes ilvBN, ilvGM and ilvIH were cloned from metsulfuron-methyl resistant strain Klebsiella sp. HR11 and metsulfuron-methyl sensitive strain Klebsiella pneumoniae MGH 78578. Homologous sequences comparison indicated that the differences in AHAS isozyme genes at amino acid levels between strain HR11 and strain MGH 78578 were mainly on the large subunits of ilvBN and ilvGM. The three AHAS isozyme genes from HR11 and MGH 78578 were ligated into the expression vector pET29a(+) and expressed in Escherichia coli BL21, respectively. The results of enzyme inhibition assay showed that only ilvBN and ilvGM from strain HR11 showed strong resistance to AHAS-inhibitor herbicides, while ilvIH from strain HR11 and ilvBN, ilvGM and ilvIH from strain MGH78578 were sensitive to AHAS-inhibitor herbicides.