Isolation & Characterization Of Butachlor Degrading Strains And Cloning Of Its N-Dealkylase Gene

Chloroacetamide herbicides are a class of pre-emergence herbicide with high efficiency and high selectivity. They are used worldwide to control broadleaf weeds and annual grasses. Butachlor and acetochlor are the two representatives of chloroacetamide herbicides and their annual consumption exceeds 10000 tons and 30000 tons, respectively. Chloroacetamide herbicides have toxicity towards fishes and butachlor was listed as L2 Carcinogens by Environmental Protection Bureau. Because of their high water solubility and long persistence in the soil, so the residues of chloroacetamide herbicides not only injure the subsequent rotation crops but also contaminate the ecological enviroment. Therefore, great concerns have been raised regarding the residues of chloroacetamide herbicides in the environment.Previous studies have proved that biodegradation is the most important method to remove the chloroacetamide herbicides in the environment, so the present study used butachlor as the target substrate and aimed at isolating bacteria capable of degrading chloroacetamide herbicides, elucidating the degradation pathway of butachlor, cloning the key genes, and thus elucidating the degradation mechanism of chloroacetamide herbicides at molecular level.1. Isolation of butachlor-degrading strains and study on its degradation characteristicsUsing butachlor as the target substrate, two enrichments designated as Y1 and Y2 were isolated by a successive enrichment method with the soil sample collected from the rice field in Changzhou of Jiangsu province. A strain named Bl was isolated from Y1 and two strains B2 and N7 were isolated from Y2. Based on the phenotypic, physiological and biochemical characteristics and the phylogenetic analysis of 16S rRNA gene sequences, strains B1 and B2 were preliminarily identified as Rhodococcus sp. The taxonomic status of strain N7 was studied in the last chapter.The degradation rate of strain B1 against 100 mg/L butachlor with five days was 100%. Strain B2 could degrade 94% butachlor within 12 h. The optimal temperature、pH for the degradation of butachlor by strain B1 and B2 were 30℃,7.0, respectively.0.1 mM Ag+, Ca2, Ni2+ and Hg2+ intensively inhibited the degradation by strain B1 and 0.1 mM Hg2+, Ag+ showed the highest inhibition effect.0.1 mM Ag+, Ba2+, Co2+, Mn2+, Hg2+ and Cu2+ inhibited the degradation intensively and 0.1 mM Hg2+ and Mn2+ showed the highest inhibition effect.0.1 mM of Mg2+ showed a certain degree of promotion on the degradation by strains B1 and B2.A metabolite was detected by GC-MS during the degradation of butachlor by strain B1、 B2 and identified as 2-chloro-N-(2’,6’-ethyl)phenyl-acetamide (CDEPA), which could not be further degraded by strain B1、B2. Thus the degradation of butachlor by strains B1, B2 was proposed as the breakdown of the C-N bond between butoxymethyl and the N atom of aniline (degradation by N-dealkylation).Strain B1 was able to degrade alachlor, acetochlor and pretilachlor, while strain B2 was only able to degrade acetochlor and butachlor. The substrate spectrum of strain B1 was broader than that of strain B2. So, strain Bland B2 have the same function to breakdown of the C-N bond between butoxymethyl and the N atom of aniline but the catalytic enzyme in the process might be different. We will study the mechanism of butachlor degradation by B1 and B2.Strain N7 could not degrade butachor and CDEPA but it coexists stablely in the enrichment culture with strain B2.The cell concentration of N7 was increased when cultivated in the medium containing butachlor after degradation by B2, therefore strain N7 can use metabolite other than CDEPA produced during the degradation of butachlor by strain B2.2. Purified the butachlor-degrading enzyme (Ch1H) from strain B1 and study on its charactersA qualitative analysis method of butachlor degradation was established according to fact that the crude extract of strain B1 could produce a transparent halo on the agarose agar by the degradation of butachlor. Ch1H was acquired by DEAE anion exchange, Q anion exchange and PAGE extraction. The enzyme was purified 185.1-fold with a 16.1% yield after the final step with a specific activity of 114.8 U/mg of protein.The molecular mass was 45 kDa. It can hydrolysis the butachlor to CDEPA. The optimal pH of Ch1H was observed to be 7.0. 1mM Hg2+, Ca2+, and Ni2+ were able to severely inhibit its activity, 1mM Fe2+ can inhibit its activity and the addition of Mg2+ increased the enzymatic activity by 1.1-fold. Ch1H could also catalyze the N-dealkylation of alachlor, acetochlor and pretilachlor. The best substrate was alachlor. The hydrolysis efficiency descended as follows:alachlor> acetochlor> butachlor> pretilachlor, indicating that the hydrolysis activities of the Ch1H decreased with the increase of the alkyl chain length. We failed to clone the gene of Ch1H using the method of N-terminal sequencing of the target protein and we will continue to do this job in future research.3. Cloning of N-dealkylase gene involved butachlor degradation in Rhodococcus sp. B2 by genome comparisonThe degradation character of strain B1 was stable by successive streaking on LB agar. But a few colonies of strain B2 was lost the ability to degrade butachlor after successive streaking on LB agar. One such mutant was designated as TB2. The genomes of B2 and TB2 were sequenced utilizing the Illumina MiSeq technology. The draft genomes of strains B2 were 6,871,517 bp in length and the total number of ORFs was 6521. The length of draft genome of strain TB2 was 6,716,155 bp and the total number of ORFs was 6411.By comparing the genomes of strains B2 and TB2 with software Omiga 2.0, a fragment (about 30 Kb in length) from scaffold 51 of strain B2 was found to be absent in the genome of mutant strain TB2. This finding was confirmed by PCR detection with the four pairs of primers distributed in different areas of the absent fragment. A gene cluster consisting of EthR EthA, EthB, EthC and EthD five genes was found by the analysis all ORFs and its encoding amino acid sequence of the missing fragment in NCBI (http://blast.ncbi.nlm.nih.gov/). EthR, encoding a regulator of AraC/Xy1S family (37KDa), shares highest similarity with the EthR from Rhodococcus ruber IFP2001 (99%). EthA, encoding a ferredoxin reductase (43KDa), shares highest similarity with the EthA from Rhodococcus ruber IFP2001 (99%). EthB, encoding a cytochrome P450 oxidase (44 kDa), shares highest similarity with the EthB from Rhodococcus ruber IFP2001 (92%). EthC, encoding a 2Fe-2S ferredoxin (11 kDa, shares highest similarity with the EthC from Rhodococcus ruber IFP2001 (99%). EthD, coding a protein of unknown function (10 kDa), shares highest similarity with the EthD from Rhodococcus ruber IFP2001 (90%). Rhodococcus ruber IFP2001 is an ETBE (ethyl tertiary butyl ether)-degrading strain, its gene cluster EthRABCD encoding a protein complex responsible for the the O-dealkylation of ETBE (breakdown C-O bond). So we deduced that the gene cluster EthRABCD in strain B2 might have the similar function and encode the enzymes for degrading butachlor through the breakdown of C-N bond between butoxymethyl and the N atom of aniline (perform the fuction of N-dealkylation).In order to identify the function of EthRABCD of strain B2, EthRABCD gene cluster (containing its native promoter) was cloned into pRESQ to construct a recombinant plasmid pQeth1, which was transformed into to mutant strain TB2. The complementation strain resumed its ability to degrade butachlor. So we think that the gene cluster EthRABCD is responsible for the N-dealkylation of butachlor in strain B2. The complementation strain TB2 (pQEthl) could degrade acetochlor and butachlor without induction. It could transform pretilachlor and alachlor to CDEPA after the induction of acetachlor.In view of the unknown function of EthD in Rhodococcus ruber IFP2001, the recombinant plasmid pQeth2 (pRESQ haboring EthRABC) was introduced into mutant strain TB2, but this process did not recover the ability of butachlor degradation. Therefore EthD was indispensable for the EthRABCD gene cluster, but its role still needed further study. To express the gene cluster EthABCD in E. coli, the recombinant plasmid pET-EthABCD, constructed by EthABCD and pET29a (+) was trasformed into the E. coli BL21(DE3) and the transformants obtained the function of degradation of acetochlor, but activity was low, this might be cuased by the codon usage bias during the gene expression.RT-PCR was carried out with EthB as the target gene in investigating its transcriptional level in the cells, strain B2 was subjected to the induction of different chloroacetanilide herbicides and glucose. In comparison with the induction glucose, the transcriptional level of EthB increased by fold of 0.73,354,33 and 1.63 with the induction of alachlor、 acetochlor、pretilachlor and butachlor, respectively. The result showed acetochlor and butachlor could induce the expression of EthB and acetochlor had the highest efficiency. The further study revealed that the transcriptional level of EthR, EthA, EthB, EthC and EthD were all induced by acetochlor and increased by the fold of 326,256,354,321 and 263, respectively.The gene cluster EthRABCD was not detected in strain B1 using PCR method. It is further proved that the gene of encoding N-dealkylase to butachlor was different in strain B1 and B2.4. Investigation on the taxonomic status of strain N7The above mentioned strain N7 can co-exist with strain B2 stablely in the enrichment liquid and it can use the metabolites generated from the degradation of butachlor by strain B2. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain N7T was a member of the genus Sphingobacterium and showed the highest sequence similarities to Sphingobacterium multivorum IAM143161 (97.49%), Sphingobacterium canadense CR11T (97.11%) and lower (< 97%) sequence similarities to all other Sphingobacterium species. Sphingobacterium sp. N7, Sphingobacterium multivorum IAM14316T, Sphingobacterium canadense CR11T(AY787820) and Sphingobacterium siyangense SY1T(EU046272) forms a branch in the phylogenetic tree. Strain N7T possessed MK-7 as the predominant ubiquinone, iso-C15:0 (25.49%), C16:0 (7.92%), iso-C17:03-OH (6.98%), and summed feature 3 (comprising C16:1ω6c and/ or C16:1ω7; 42.08%) as the major fatty acids. The DNA G+C content was 40.9 mol ± 0.5 mol%. Strain N7T showed low DNA-DNA relatedness with Sphingobacterium multivorum IAM14316T(21%), Sphingobacterium canadense CR11T (15%), was lower than 70%(the interspecies threshold). Based on the the results of polyphasic analysis, strain N7T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium changzhouense N7T(= CCTCC AB 2012100T= KACC 16854T) was proposed.