Molecular Mechanism Of The Upstream Catabol Ic Pathway Of Buprofezin In Rhodococcus Qingshengii YL-1 And Isolation Of A Buprofezin-degrading Strain With Stable Phenotype

Buprofezin is an insect growth regulators,whose pesticidal action lies in its inhibition of chitin biosynsthesis and cell metabolism,therefore interfering with the normal molting and developmental metamorphosis of target pests.It has been applied extensively on rice,tea,potatoes,fruits,cotton,vegetables and exhibits a persistent larvicidal action against the planthopper,leafhopper and aleyrodid.Due to its widespread use,buprofezin residue has been frequently detected in food,soil and drinking water.Toxicity studies have shown that buprofezin is toxic to some aquatic organisms and non-target insects.Meanwhile,buprofezin poses a potential threat to human health,so the elimination of buprofezin residues in agricultural products and the environment has become an urgent problem needed to be addressed.Microbial degradation plays an important role in the elimination of buprofezin and microbial remediation is an ideal means of removing buprofezin residues from the environment.However,the molecular mechanism of microbial metabolism of buprofezin is unclear.In addition,there is a lack of degradation phenotype stable strain of the strain sources that limited the microbial remediation of buprofezin residues.Previous studies have shown that Rhodococcus qingshengii YL-1 could utilize buprofezin as the sole carbon source for growth,and can significantly accelerate the elimination process of buprofezin in contaminated soil,implying the potential use of this strain in bioremediation.However,the buprofezin-degrading phenotype of strain YL-1 is unstable when it grows in nutrient-rich medium.In order to lay a solid foundation of microbial remediation of buprofezin residues,the aims of this study are as follows:First,to clarify the upstream catabolic pathway and molecular mechanism involved in buprofezin degradation;Second,to construct or isolate buprofezin degrader with stable degradation phenotype.1 Identification of the upstream catabolic pathway of buprofezinTo provide reference for the cloning of the degradation genes,the upstream catabolic pathway of buprofezin by strain YL-1 was identified first.To identify the upstream catabolic pathway of buprofezin in strain YL-1,the intermediate metabolites that are made before the production of 2-BI were analyzed using ultra-high-performance liquid chromatography tandem mass spectrometry(UHPLC-MS).Three new compounds were detected.Accordingly,the upstream catabolic pathway of buprofezin was proposed:the benzene ring of buprofezin was initially dihydroxylated into buprofezindihydrodiol(BF-DD),which was subsequently dehydrogenated to generate dihydroxybuprofezin(DHBF);DHBF was then subjected to aromatic ring cleavage to give rise to aromatic ring-cleavaged dihydroxybuprofezin(RCDB);finally,2-BI was released from RCDB through the breaking of the amide bond.2 Prediction of buprofezin-catabolic genes via comparative genomic analysisThe degradation phenotype of strain YL-1 is unstable,some colonies lost the ability to degrade buprofezin after 3 rounds of continuous transfer in LB broth,with one such mutant strain designated YL-0.In addition,mutant strain YL-0 exhibited an obviously shorter lag phase than did the wild type YL-1 when grown in LB broth.The complete genome of strain YL-1 was 7,594,958 bp in length and revealed six replicons,consisting one circular chromosome,three circular plasmids(pYLC1,pYLC2,pYLC3)and two putative linear plasmids(pYLL1 and pYLL2).The results of the sequence comparison and the PCR amplification showed that,compared with strain YL-1,mutant strain YL-0 lost a fragment of approximately 200 kb that is located on plasmid pYLC1 of strain YL-1.Based on the upstream catabolic pathway proposed above,the candidate genes,designated bfzBA3A4A1A2C,that are involved in the degradation of buprofezin were predicted from the missing fragment.Gene cluster bfzA3A4A1A2 was predicted to encode a multicomponent Rieske non-heme iron dioxygenase(RHO).BfzAl and BfzA2 exhibit similarities to the corresponding α(35-38%)and β(45～47%)subunits of some aromatic ring-hydroxylating dioxygenases,respectively.BfzA3 and BfzA4 show similarities to the corresponding[2Fe-2S]-type ferredoxin(48～50%)and glutathione reductase(GR)type ferredoxin reductase(32～34%)components of the electron transport chain(ETC)of RHOs,respectively.All of these analyses indicated that bfzA3A4A1A2 is most likely responsible for the dioxygenation of buprofezin to generate BF-DD.The product of bfzB exhibits 24～46%identities to the aryl-alcohol dehydrogenase,which indicates that BfzB is probably involved in the dehydrogenation of BF-DD to give rise to DHBF.BfzC shows 30～43%identities to extradiol ring-cleavaged dioxygenases,implying that BfzC is likely responsible for the aromatic ring cleavage of dihydroxybuprofezin.3 Function verification revealed that bfzBA3A4A1A2C is responsible for the degradation of buprofezinTo verify bfzBA3A4A1A2C cluster is involved in the upstream catabolic pathway of buprofezin in strain YL-1,the plasmid pREBAC containing bfzBA3A4A1A2C,was introduced into mutant strain YL-0.The plasmid pREBAC restored the ability of mutant strain YL-0 to utilize buprofezin as the sole carbon source.These results indicated that gene cluster bfzBA3A4A1A2C is responsible for the dihydroxylation,dehydrogenation,and aromatic ring cleavage of buprofezin;in addition,the genes responsible for breaking the amide bond of aromatic ring-cleaved dihydroxybuprofezin and for the downstream catabolic pathway have not been lost in the mutant strain YL-0.To further validate the roles of each gene in bfzBA4A3A1A2C,unmarked deletion strains of these genes were individually constructed.Knockout of bfzA1,bfzA2 and bfzA3 resulted in the inability of mutant strains YL-1△bfzA1,YL-1△bfzA2 and YL-1△bfzA3 to degrade buprofezin.The complemented strains YL-1△bfzA1(pREA1),YL-1△bfzA2(pREA2)and YL-1△bfzA3(pREA3)regained their abilities to degrade buprofezin.Mutant strain YL-1△bfzA4 showed a decreased ability to degrade buprofezin compared with its wild type.These results implied that bfzA1A2A3A4 is responsible for the initial step of buprofezin degradation.Deletion of bfzB led to the accumulation of BF-DD,indicating that bfzB is responsible for the dehydrogenation of BF-DD.The accumulation of dihydroxybuprofezin was observed in bfzC knockout mutant YL-1△bfzC.The complemented strain YL-1△bfzC(pREC)regained its ability to degrade buprofezin,indicating bfzC is responsible for the ring-cleavage of DHBF.Collectively,the proposed upstream catabolic pathway of buprofezin degradation has been proven at the molecular level.In vitro validation of BfzA1A2A3A4 was carried out.BfzA1A2 and BfzA3 were successfully purified to homogeneity as His-tagged recombinant proteins using Escherichia coli Rosetta(DE3)pLacI as expression host,while bfzA4 was expressed as inclusion body even after optimization.Afterwards,bfzA4 was expressed in R.erythropolis L88,despite only a small fraction of BfzA4 was present at the supernant of cell extract.The enzyme mixture with a combination of BfzA1A2,BfzA3,and BfzA4 exhibited weak yet detectable enzymatic activity against buprofezin,evidenced by the formation of BF-DD.The above results confirmed the functionality of multicomponent BfzA1A2A3A4 at enzymatic level.The phylogenetic analysis of BfzAl,comparing it with the large subunits of 60 characterized RHOs,indicated that BfzAl belongs to the group involved in aromatic ring hydroxylation.However,BfzA1 formed a new branch within this group.In another classification system suggested by Kweon et al.,in which the RHOs are classified into five groups based on the analysis of the oxygenase and ETC components of the RHOs.According to this classification system,BfzA1 A2A3A4 falls into the type IV RHOs,whose members generally contain a hetero-oligomer-type oxygenase(αnβn),a[2Fe-2S]-type ferredoxin,and a glutathione reductase(GR)-type reductase.BfzAl also forms a new branch within this group,indicating it is a new member of the type IV RHOs.Since bfzBA3A4A1A2C was homologous with the genes responsible for the upstream catabolic pathway of biphenyl,the sequence of BfzA1 was compared with those of the large subunits of the characterized biphenyl dioxygenases.The large subunits of biphenyl dioxygenases form a clade,while BfzAl occupied a separate clade.Substrate spectrum experiment showed that strain YL-1 could also degrade biphenyl and its analogs such as flavone,flavanone and bifenthrin,and yellow-colored(for biphenyl,flavanone and bifenthrin)or orange-colored(for flavone)intermediate metabolites appeared during metabolism.By contrast,mutant strain YL-0,YL-1△bfzAl,and YL-1△bfzC could not.The aromatic ring-cleaved products of the four substrates were detected using UHPLC-MS analysis,the results of which indicated that products of bfzBA4A3A1A2C could also catalyze the dihydroxylation,dehydrogenation and aromatic ring cleavage of biphenyl,flavone,flavanone and bifenthrin.In addition,the bfzBA4A3A1A2C genes were proven to be organized in one transcriptional unit via real-time PCR(RT-PCR)and its expression is not induced by buprofezin or biphenyl.4 Isolation and identification of buprofezin-degrading strain Rhodococcus sp.D-6One strain,designated Rhodococcus sp.D-6,which could grow with buprofezin as the sole carbon source,was isolated from the paddy soil contaminated with buprofezin in Dafeng District,Yancheng City,Jiangsu Province,by enrichment method.When the initial concentration of buprofezin was set at 50 mg-L-1,over 95%of buprofezin was degraded by strain D-6 within 48 hours after incubation.The optimal temperature and pH for the degradation of buprofezin by strain D-6 are 30℃ and pH 7.0,respectively.The detection of the intermediate metabolite 2-BI and the amplification of the corresponding products of bfzBA3A4A1A2C in strain D-6 revealed strain YL-1 and D-6 shared the same upstream catabolic pathway of buprofezin degradation.The degradation phenotype of buprofezine degradation phenotype of strain YL-1 is not stable as approximately 15%of cells lost the ability to degrade buprofezin after 10 rounds of transfer in LB medium;while strain D-6 is not ready to lost the degradation ability,evidenced by the fact that strain D-6 maintained the degradation phenotype even after 10 rounds of transfer in LB medium.In summary,this study elucidated the upstream catabolic pathway of buprofezin degradation and the corresponding molecular mechanism,therefore enhanced our understanding in microbial degradation of buprofezin.Second,the gene cluster bfzBA3A4AlA2C is responsible for the degradation of buprofezin,biphenyl and its analogues,and has potential application value.Finally,the degradation phenotype of the newly isolated strain D-6 is stable,which provided excellent strain resources for microbial elimination of buprofezin residues.