Study On The Molecular Mechanism Of THF Degradation In Cupriavidurans Metallidurans ZM02

Tetrahydrofuran(THF)is a typical representative of oxygen heterocyclic compounds.It is widely used as an important basic chemical feedstock for organic synthesis,an excellent polar solvent,and a promoter of chemical reactions in the pharmaceutical and chemical industries.THF has toxic and carcinogenic activity.It is classified as a refractory organic contaminant and is widely distributed in underground water.At present,the molecular mechanism of microbial degradation of THF is not clear yet.The reported THF-degrading gene clusters are all derived from Gram-positive bacteria.The THF-degrading gene clusters of Gram-negative bacteria need to be explored,and the research on related genes and enzymes involved in the downstream metabolic pathways of THF is lacking.In this study,we isolated a Gram-negative THF-degrading bacterium from environmental samples contaminated by THF for a long time.The potential THFdegrading gene clusters were discovered through genome and transcriptome analysis,and the gene function was verified using molecular biology methods.This study aims to reveal the degradation mechanism of THF in Gram-negative bacteria.The main research results are as follows:(1)Analysis of degradation characteristics of Gram-negative THF-degrading bacterium and identification of metabolites during THF degradation.A new Gram-negative THF-degrading bacterium,designated Cupriavidurans metallidurans ZM02,was isolated from activated sludge of sewage treatment plant contaminated by THF for a long time.Strain ZM02 was able to aerobically utilize THF as the sole carbon and energy source and could tolerate a maximum THF concentration of 120 mM.20 mM THF could be completely degraded in 168 h,and 2.5 mM phenol could be completely degraded in 15 h by strain ZM02.Meanwhile,strain ZM02 had a wide spectrum of substrates and could utilize intermediate metabolites of THF(including 2-hydroxytetrahydrofuran,?-butyrolactone,and succinate),3-hydroxybutyrate,phenol,catechol,1,4-butanediol,benzene,toluene,benzoic acid,and acetic acid as the sole carbon source.The intermediate metabolites 4-hydroxybutyrate,succinate,and the by-product acetic acid were detected and identified by GC and GC-MS analyses during the THF degradation process.(2)Genome and transcriptome analyses revealed the potential novel THF-degrading gene cluster.The genome of strain ZM02 is 6,258,398 bp in length,containing two chromosomes and a circular plasmid.Transcriptome analysis revealed that there is a potential THF degradation gene cluster dmpKLMNOPcat A on the chromosome.The gene cluster dmpKLMNOP encodes multi-component THF hydroxylase,and cat A encodes catechol 1,2-dioxygenase.The amino acid sequence identity between the THF hydroxylase gene cluster dmpKLMNOP in strain ZM02 and other reported THF-degrading gene clusters thm ADBC and prmABCD in Gram-positive bacteria were both lower than 41%.The THF hydroxylase Dmp KLMNOP belongs to the soluble di-iron monooxygenases(SDIMOs)and has a close relationship with phenol monooxygenase(Group 2).(3)Cloning and functional identification of the novel THF hydroxylase gene cluster dmpKLMNOP.RT-q PCR verified that expression of the THF hydroxylase gene cluster was significantly up-regulated when using THF and phenol as the inducing substrates.The ?dmpL,?dmpN,?dmpP,and ?dmpR were successfully obtained through gene knockout and lost the ability to degrade THF and phenol,but the utilization of intermediate metabolites of THF(2-hydroxytetrahydrofuran,?-butyrolactone),and phenol(catechol)was not influenced,indicating that the loss of THF hydroxylase function only affects the conversion of THF to 2-hydroxytetrahydrofuran in the first step.At the same time,different recombinant vectors harboring all or part of the THF hydroxylase gene cluster were constructed and expressed heterologously in the non-degrading bacterium C.pinatubonensis JMP134.It was found that the THF hydroxylase encoded by dmpKLMNOP was involved in the degradation of THF,and the gene cluster dmpKLMNO is indispensable for the catalytic function of THF hydroxylase.In addition,enzyme activity analysis confirmed that the THF hydroxylase encoded by dmpKLMNOP could use NADH or NADPH as an electron donor to catalyze the degradation of THF and phenol.In summary,a new THF degradation gene cluster dmpKLMNOP was found in the Gram-negative bacterium C.metallidurans ZM02,which encodes THF hydroxylase involved in catalyzing the hydroxylation of THF to 2-hydroxytetrahydrofuran.(4)Cloning and functional identification of catechol 1,2-dioxygenase gene cat A.The expression of cat A1,a gene encoding catechol 1,2-dioxygenase,located near the THF hydroxylase gene cluster dmpKLMNOP,was significantly up-regulated under the induction of THF.Strain?cat A1 could degrade THF and its intermediate metabolites(2-hydroxytetrahydrofuran,?-butyrolactone),and phenol and its intermediate metabolites catechol.It was found that there is another gene catA2 also encoding catechol 1,2-dioxygenase in the benzoic acid degradation gene cluster of strain ZM02 based on the genome annotation analysis.The amino acid sequence identity between catA2 and cat A1 reached 63%.It was speculated that CatA2 has an alternative function to compensate that in strain ?cat A1.In addition,the gene knockout and gene complementation results confirmed that the 2,3-dihydroxyphenylpropionate 1,2-dioxygenase encoded by mhp B in Escherichia coli BL21 was involved in the conversion of THF intermediate metabolite 2-hydroxytetrahydrofuran to 4-hydroxybutyrate.In this study,the expression plasmids p ET28 acat A1 and p ET28a-catA2 were constructed and expressed in strain E.coli BL21 ?mhp B.Both heterologous expression strains could successfully convert 2-hydroxytetrahydrofuran to 4-hydroxybutyrate.In summary,the results indicated that cat A1 and catA2 are both involved in the catalytic process of 2-hydroxytetrahydrofuran to 4-hydroxybutyrate in the THF metabolic pathway in the Gram-negative bacterium C.metallidurans ZM02.(5)The potential reasons why the phenol-degrading bacteria cannot degrade THF alone.THF hydroxylase and phenol monooxygenase which catalyzes the degradation of phenol are highly homologous,and the gene clusters of the two monooxygenases are similar in composition and structure.Representative phenol-degrading bacteria C.pinatubonensis JMP134,Alcaligenes faecalis JQ135 and Sphingomonas melonis TY could grow with phenol as the sole carbon source,but could not grow with THF as the sole carbon source.When phenol was used as an inducing substrate,C.pinatubonensis JMP134 and A.faecalis JQ135 could co-metabolize THF,but S.melonis TY still could not co-metabolize THF.C.pinatubonensis JMP134 could grow with intermediate metabolite 2-hydroxytetrahydrofuran as the sole carbon source,while A.faecalis JQ135 and S.melonis TY could not grow with 2-hydroxytetrahydrofuran as the sole carbon source but could biotransform 2-hydroxytetrahydrofuran to 4-hydroxybutyrate.When phenol was used as an induction substrate,the expression of dmpN encoding the large subunit of phenol monooxygenase and dmpR encoding the transcriptional regulatory protein in the three representative phenol-degrading bacteria were all significantly up-regulated.The above results showed that a variety of factors affect the degradation of THF by phenol-degrading bacteria containing similar THF-degrading gene clusters,including THF cannot induce the expression of THF-degrading gene clusters as the sole substrate;the function of phenol monooxygenase to degrade THF is missing;the enzyme involved in catalyzing the downstream metabolic pathway of THF is missing.In this study,we found a new THF-degrading gene cluster in Gram-negative THF degrading bacteria,and verified the function of the THF-degrading gene cluster.The potential of representative phenol-degrading bacteria to co-metabolism THF and the reasons for the inability to degrade THF alone were analyzed.The research is of great significance to reveal the diversity of microbial degradation of THF and to further explore the evolutionary laws of THF-degrading gene clusters.