Pathway And Enzyme Responds Mechanism On The Biodegradation Of Diethyl Phthalate By Sphingobium Yanoikuyae SHJ

Groundwater is one of the valuable fresh water resources and one of the important drinking water sources for human.However,with the rapid development of industry and agriculture,groundwater has suffered more and more serious pollution.Phthalate esters（PAEs）are main organic pollutants in groundwater,and PAEs in groundwater have great potential threats to human health and ecological environment.Microbial remediation of PAEs contaminated groundwater has broad application prospects.The study of the biodegradation pathway of PAEs provides a powerful basis for clarifying the migration and transformation of PAEs in groundwater environment.The enzymology response and mechanism of microbial degradation of PAEs in groundwater can provide theoretical support for the remediation of contaminated groundwater.Diethyl phthalate（DEP）is a one of the representative polluntants of PAEs.A DEP-degrading bacterium Sphingobium yanoikuyae SHJ isolated from the shallow aquifer sediments of the Jianghan Plain was used to study the biodegradation kinetics of DEP under aerobic and simulated shallow aquifer oxygen-limited conditions by HPLC-HRMS.The intermediates during the DEP degradation process were identified and the DEP degradation pathways were proposed.The whole genome sequence was obtained by using Illumina Miseq sequencing platform and PacBio RS Ⅱ sequencing platform,and the protease genes related to DEP degradation were obtained by gene annotation analysis.The differential proteins in the presence or absence of DEP were studied by the method of combining the quantitative analysis of protein and mass spectrometry with the relative and absolute quantitative equivalent heterotopic labeling technology（iTRAQ）,and the proteases involved in DEP degradation were speculated.A series of column separation techniques were used to isolate and purify the key enzymes in the degradation pathway to further determine the key proteases for DEP degradation.Through the above research,the following conclusions were obtained:（1）Isolation and identification of strain SHJ and its degradation kinetics of DEPBy enrichment culture,the DEP-degrading strain named SHJ was isolated from the shallow aquifer sediments of the Jianghan Plain.The 16S rDNA gene sequences of Strain SHJ（GenBank accession number JFFT01000000）showed a high similarity with Sphingobium yanoikuyae ATCC 51230.The strain can degrade DEP by using DEP as sole carbon source under oxygen-limited conditions in simulated shallow aquifer（17 ^oC,static,dark and oxygen-limited）.The DEP degradation process by Sphingobium yanoikuyae SHJ accords with first-order kinetic equation.Under aerobic conditions,the degradation rate constant k of DEP by SHJ is 0.8139 h^-1 and the degradation half life is0.85 h.Under the simulated shallow aquifer（oxygen-limited）environment,the degradation rate constant k of DEP by SHJ is 0.1534 h^-1 and the degradation half life is4.52 h.（2）Biodegradation pathway and mechanism of Sphingobium yanoikuyae SHJ on DEPIn the DEP bidegradation process by Sphingobium yanoikuyae SHJ,high-performance liquid chromatography tandem high-resolution mass spectrometry（HPLC-HRMS）was used to capture and analyze the intermediates.Four intermediates of DEP were identified by SIM or MS/MS,including ethyl methyl phthalate（EMP）,monoethyl phthalate（MEP）,monomethyl phthalate（MMP）and phthalic acid（PA）.According to the changes of DEP and these intermediates with time,it was proposed that two DEP degradation pathways must be present in Sphingobium yanoikuyae SHJ.The first was DEP→MEP→PA through a sequential hydrolysis process.The second was DEP→EMP→MEP→MMP→PA through a combination of demethylation or trans-esterification with methanol and hydrolysis processes.Under the simulated shallow aquifer（oxygen-limited）conditions,EMP,MEP and MMP can be degraded sequentially,but PA can not further degrade.Under optimum aerobic conditions,DEP and its four intermediates can be degraded rapidly.Therefore,it is suggested that in the shallow aquifer environment（oxygen-limited）,Sphingobium yanoikuyae SHJ requires oxygen addition to thoroughly degrade DEP.（3）Genome-wide sequencing analysis of Sphingobium yanoikuyae SHJGenome-wide sequencing of Sphingobium yanoikuyae SHJ was carried out using Illumina Miseq sequencing platform and PacBio RS Ⅱ sequencing platform.And genetic prediction and functional annotation of the genome sequences were carried out to identify genes related to PAEs degrading enzymes.The total number of fragments obtained by whole genome sequencing of the strain SHJ is 438,236,and the total length of the fragments is 1,800,913,400.The whole genome sequence is a complete sequence with a total length of 5,669,383bp and a GC content of 64.23%,including a cyclic chromosome（Chr）with a length of 5,258,921bp and a GC content of 64.36%（Chr）and additional two plasmids（pSES220 and pSES189）with two lengths of 189,186bp and220,039bp and two GC content of 61.43%and 63.68%respectively.Functional annotation of SHJ revealed a total of 5,402 genes,with 5,183 protein-encoding genes,143pseudo genes,12 rRNA genes,61 tRNA genes and 3 other non-coding RNA genes.Based on genome annotation,5,033 open readings were obtained.Whole genome analysis revealed 44 genes encoding for acylesterase,esterase or carboxylesterase,and alpha or beta hydrolase,probably involved in hydrolysis of PAEs to the corresponding monoesters and phthalate acid,and 13 genes encoding hydrolases and esterases have homologous amino acid sequences with PAEs metabolizing enzymes in nr database.Also a region with size of about 6.9 kb comprised of seven ORFs（phtRCBAaDAcAd）,which located on the smaller plasmid pSES189,was presumed to be used in the transformation of PA into protocatechuic acid,and the gene composition in the gene cluster is highly similar to that of the gene cluster pht（phtBAaAbAcAdCR）that has been reported in the plasmid of the PA degrading bacterium Arthrobacter keyseri 12B.The results help to lay a foundation for the further study of the key enzymes in the metabolic process.（4）Proteomic analysis of DEP biodegradation enzymes by Sphingobium yanoikuyae SHJDifferential proteomics was used to analyze the differences in the protein expression of Sphingobium yanoikuyae SHJ in the extracellular enzymes and intracellular enzymes produced at different incubation times（24 h was chosen for the early stage of DEP degradation and 96 h for the late stage）by DEP and glucose as the sole carbon source,respectively,and to speculate the important enzymes related to the biodegradation of DEP and their response to the DEP metabolic process.The expression of these enzymes in the presence of DEP was more abundant than glucose.Using differential corrected protein p-value（p<0.05,fold>1.2）,727 functional predictive proteins were identified from all comparison groups.Statistics of all differentially expressed proteins,which were over 2 times over the comparison groups,were lower than 0.5 times.Compared with glucose as carbon source,Sphingobium yanoikuyae SHJ grew with DEP as carbon source,and the total number of differential proteins from extracellular enzyme produced at the early stage of degradation was 58（of which 41proteins were up-regulated,mainly involving extracellular hydrolase,methyltransferase and carbon carbon lyase）.The total number of differential proteins from intracellular was 119（of which 26 were up-regulated,mainly involving intracellular hydrolases）.The total number of differential proteins from extracellular enzyme produced in the late stage of degradation was 71（of which 43 were mainly involving extracellular hydrolase,methyltransferase,transferase,carbohydrate catalytic enzyme,carbon carbon lyase）,and the total number of differential proteins from intracellular enzyme was 162（of which 89were up-regulated,mainly involving intracellular hydrolase,methyltransferase,transferase and carbohydrate catalytic enzyme）.Compared to the early stage,the total number of differential proteins from extracellular produced by Sphingobium yanoikuyae SHJ in the late stage of DEP degradation was 119（of which 64 proteins were up-regulated,mainly involving extracellular hydrolase,methyltransferase,transferase,carbohydrate catalyzed enzyme and carbon carbon lyase）,and the total number of differential proteins from intracellular enzyme was 222（of which 125 were upregulated,mainly involving intracellular hydrolases,transferases,and carbohydrate catalyzed enzymes）.In addition,the expression of some membrane protein,lipoprotein and pili protein increased obviously in the early stage of DEP degradation,while the transport protein,chaperone protein and mitotic protein were significantly up-regulated in the late stage of DEP degradation.Through bioinformatics analysis of related protein functions,it was found that the enzymes related to DEP degradation were obviously induced or expressed significantly.These differentially expressed proteins were involved in the metabolic pathway of DEP biodegradation by Sphingobium yanoikuyae SHJ.The study revealed the proteomic and metabolic changes of strain Sphingobium yanoikuyae SHJ exposed to different nutritional environments.（5）Isolation,purification and identification of key enzymes of DEP biodegradation by Sphingobium yanoikuyae SHJIt was established on the basis of enzyme activity determination and its enzymological properties to isolate and purify the DEP biodegradation enzyme.Four purified bands（Ⅰ、Ⅱ、Ⅲ、Ⅳ）were finally purified,and their protein identification and function prediction were carried out.The final purification multiplier of band Ⅰ was 1.21and the apparent molecular weight was 58.50 kD,which had the activity of transferase,the activity of dioxygenase and the function of transporter;the final purification multiplier of band Ⅱ was 1.21 and the apparent molecular weight was 35.75 kD,which had isomerase activity,hydrolase activity and transferase activity.The purification multiplier of band Ⅲ was 1.19 and the apparent molecular weight was 68.94 kD,it is a carbon carbon lyase,which catalyzes the breaking of the C-C bond.The final purification multiplier of the band Ⅳ was 1.19 and the apparent molecular weight was62.76 kD,and it has the activity of transferase.The results of DEP biodegradation pathway,genomics of degrading bacteria and proteomics of degrading enzymes were also studied.The results showed that the transferase from band I and band IV were involved in demethylation and transesterification of DEP,thus playing an important role in the transformation of DEP degradation from DEP→EMP→MEP→MMP→PA.The ring cracking catalysis of dioxygenase from band I did not play a role in the biodegradation process of DEP under the condition of simulated shallow aquifer（oxygen-limited）environment.It was speculated that it was mainly involved in the further degradation of PA under aerobic conditions.The protease of band Ⅱ,which has isomerase activity,hydrolase activity and transferase activity,plays a key role in the process of DEP metabolism.It was possible to participate in the two metabolic process of DEP,the first was the sequential hydrolysis of DEP to produce MEP and PA（DEP→MEP→PA）,the second was the demethylation or transesterification and hydrolysis of DEP from DEP→EMP→MEP→MMP→PA.The carbon carbon lyase of band Ⅲ is different from the hydrolysis reaction.It was speculated that the enzyme catalyzed the demethylation of DEP.Combining the results of previous studies on metabolic pathways,genomics and proteomics of DEP,the results showed that these four purified band proteases and their predictive functions indicate that the strain Sphingobium yanoikuyae SHJ has the hydrolysis reaction of DEP,the action of demethylation and the transesterification process in the degradation pathway of DEP under the simulated shallow aquifer（oxygen-limited）environment,and the three can be used as the mechanism of microbial degradation of DEP.Moreover,the encoding gene sequences of these purified degrading enzymes are in the whole genome sequence of strain Sphingobium yanoikuyae SHJ,indicating that the degradation function is encoded and expressed by the genes of the strain Sphingobium yanoikuyae SHJ,which will lay the foundation for the later construction of more effective microbial degradation engineering strains.The characteristics of this topic are:firstly,a new DEP biodegradation pathway in simulated aquifer environment was obtained.Secondly,a PA degrading gene cluster（phtRCBAaDAcAd）was found in the smaller plasmid of the Sphingobium yanoikuyae SHJ.Thirdly,the mechanism and gene coding sequence of DEP degrading key enzymes in Sphingobium yanoikuyae SHJ were revealed by whole genome sequence analysis,differential proteomics and isolation,purification and identification of the key enzymes.In this paper,the DEP biodegradation process by Sphingobium yanoikuyae SHJ was explained from the DEP microbial degradation pathway,the whole genomics of the Sphingobium yanoikuyae SHJ and proteomics of the degrading enzyme system.These three aspects are closely related and mutually corroborated,and can better reveal the biodegradation mechanism of DEP in the shallow aquifer environment.And the attempt to build more effective microbial degradation plasmids in the later period will have very important application value and innovation,at the same time,it will promote the process of using bioremediation technology to control PAEs pollution environment.