| Dimethylphenols(DMPs)contain six isomers,including 2,3-DMP,2,4-DMP,2,5-DMP,2,6-DMP,3,4-DMP and 3,5-DMP.They are common aromatics in nature and chemical intermediates widely used in chemical industry.For example,2,6-DMP can be used to synthesize the plastic polyphenylene oxide.After the wastewater containing dimethylphenols flows into the water or soil,it will seriously endanger the survival of aquatic organisms and pose a potential threat to human health.Because microorganisms are the main force of DMPs degradation in nature,the microbial degradation mechanism of DMPs has attracted extensive attention.Compared with the other five DMPs,the research on the microbial degradation of 2,6-DMP is limited,including few degradation bacteria,the incompleted metabolic pathway,and the unknown genetic and biochemical background.In order to solve the above problems,this paper aims to isolate 2,6-DMP-degrading microbes,identify the complete metabolic pathway of 2,6-DMP,and characterize the molecular mechanism of microbial metabolism of 2,6-DMP,providing strain and gene resources for microbial remediation of 2,6-DMP-polluting environment.In addition,the 2,6-DMP-degrading bacterium can also convert 2,3,6-trimethylphenol(2,3,6-TMP)into vitamin E synthesis precursor 2,3,5-trimethylhydroquinone(2,3,5-TMHQ),so the related degradation genes have potential application value in the synthesis of vitamin E precursor.1.Isolation and metabolic pathway analysis of a 2,6-DMP-degrading bacteriumA strain B5-4 which could grow with 2,6-DMP as the sole carbon source was isolated by enrichment culture.It was identified as Mycobacterium neuroarum through morphological,physiological and biochemical characteristics,and genomic alignment together with the analysis of phylogenetic trees based not only on 16 S r RNA gene sequences but also on alignment of concatenated amino acid sequences from 13 proteins with housekeeping function with the related species.A series of intermediate metabolites were identified by high performance liquid chromatography and liquid chromatography-mass spectrometry.Then,a metabolic pathway of 2,6-DMP in M.X neoaurum B5-4 was proposed,in which 2,6-DMP was initially para-hydroxylated to2,6-dimethyl-hydroquinone(2,6-DMHQ),which was then meta-hydroxylated to2,6-dimethyl-3-hydroxy-hydroquinone(2,6-DM-3HHQ);2,6-DM-3HHQ was then subjected to produce 2,4-dimethyl-3-hydroxymuconic acid(2,4-DM-3HMA)by ortho-cleavage,which was further converted to citraconate,and subsequently enters the TCA cycle.2,6-DMHQ and 2,6-DM-3HHQ would also be partially oxidized to the corresponding benzoquinone.In addition,a short delay period was captured in the initial stage of 2,6-DMP degradation by strain B5-4,which would be disappeared in the 2,6-DMP degradation by 2,6-DMP precultured cells,indicating that the transcription of some key genes responsible for 2,6-DMP degradation might be induced by substrates or its intermediates.Zebrafish was used as a living model to preliminarily evaluate the bioremediation capability of strain B5-4.The results showed that strain B5-4 has the potential to bioremediate the 2,6-DMP polluted water,and could relieve the toxic effect of 2,6-DMP on zebrafish.In addition to 2,6-DMP,strain B5-4 could partially transform 2,3-DMP and2,5-DMP,and catalyze 2,3,6-TMP to 2,3,5-TMHQ as well.2.The flavin dependent monooxygenase system MpdAB is responsible for the first step of para-hydroxylation of 2,6-DMPA 2,6-DMP degradation deficient strain B5-4M was successfully obtained by continuous subculture.Through comparative genomic analysis and transcriptomic analysis,a gene cluster mpdACDEFB was predicted,which might be involved in the degradation of 2,6-DMP.MpdA and MpdB were predicted to encode oxygenase and reductase respectively in a flavin dependent monooxygenase system,which could be responsible for hydroxylation of 2,6-DMP.A predicted dioxygenase MpdG locating upstream of the above gene cluster was likely to catalyze the cleavage of 2,6-DM-3HHQ.Similarly,a potential regulatory protein MpdR was predicted to be a transcriptional regulator of Gnt R family,which might be responsible for the transcriptional regulation of some genes involved in 2,6-DMP degradation.Moreover,the above mentioned 2,6-DMP-degrading genes were significantly up-regulated at the transcriptional level(34-to 111-folds)under the induction of 2,6-DMP.The mutant B5-4M carrying mpdA or mpdAB regained the ability to transform 2,6-DMP to 2,6-DMHQ.However,they could not further convert 2,6-DMHQ to 2,6-DM-3HHQ.This result showed that the recognition of oxygenase MpdA to its partner reductase is not specific,and some other reductases in strain B5-4M could also support the catalytic activity of MpdA.The insertion inactivation and complementarity of mpdA in strain B5-4 showed that mpdA is the sole gene responsible for transforming 2,6-DMP into 2,6-DMHQ in strain B5-4.At the same time,purified MpdAB hydroxylated 2,6-DMP with NADH and FAD as cofactors.High performance liquid chromatography,liquid mass spectrometry and nuclear magnetic resonance analysis also confirmed that MpdAB was responsible for the para-hydroxylation of 2,6-DMP to 2,6-DMHQ.In addition,the data of genetic and in vitro biochemical experiments showed that MpdAB can not only catalyze 2,6-DMP,but also hydroxylate 2,3,6-trimethylphenol(2,3,6-TMP)to 2,3,5-trimethylhydroquinone(2,3,5-TMHQ).In industry,2,3,5-TMHQ is one of the two precursors for the synthesis of vitamin E.At present,2,3,5-TMHQ is produced mainly through chemical catalysis.In this work,we constructed a whole cell catalytic system based on MpdAB.Using strain B5-4M and Escherichia coli BL21(DE3)as hosts,we preliminarily evaluated the application ability of MpdAB in the catalytic production of 2,3,5-TMHQ.The results showed that the low expression levels of MpdA and2,3,6-TMP cytotoxicity limit the efficiency of whole-cell catalysis.3.MpdD and MpdAB are responsible for the second step of meta-hydroxylation of2,6-DMP2,6-DMP was hydroxylated twice to produce 2,6-DMHQ and 2,6-DM-3HHQ by Rhodococcus qingshengii YL-1 harboring mpdACD(wild strain YL-1 could not degrade2,6-DMP;Rhodococcus is closely related to Mycobacterium,and that is used as the heterologous expression host of mpdcluster),while strain YL-1 carrying mpdA was proved to be responsible for the first step of hydroxylation of 2,6-DMP to produce 2,6-DMHQ.Unexpectedly,the data of the truncated mpdcluster and in vitro biochemical experiments showed that the maleylpyruvate isomerase MpdD together with MpdAB catalyze the second step of meta-hydroxylation of 2,6-DMP(converting 2,6-DMHQ to 2,6-DM-3HHQ).It is worth noting that this catalytic phenomenon has not been found in previous reports.We speculated that 2,6-DMHQ is first isomerized by maleylpyruvate isomerase MpdD to its isomer 2,4-dimethyl-1,3-hydroquinone,in which the hydroxyl group in para position of 2,6-DMHQ was transferred to meta position,and then para-hydroxylation is carried out by MpdAB to finally form 2,6-DM-3HHQ.This catalytic mechanism has not been reported,and the specific process needs to be further studied in the future.4.Preliminary study on the function of MpdR and MpdGTranscriptional analysis showed that mpdACDEFB is cotranscribed as a single operon.The transcriptional initiation sites of mpdACDEFB cluster and mpdR gene promoter were identified by 5’-RACE;The repressive regulation mechanism of MpdR was preliminarily explored,and MpdR would not only combine with the promoter of mpdACDEFB cluster,but also with its own promoter.The dioxygenase MpdG is predicted to be responsible for catalyzing the cleavage of 2,6-DM-3HHQ.Since there is no commercial standard of2,6-DM-3HHQ,mpdG was connected to mpdACDEFB.The resulting cells could effectively prevent the accumulation of 2,6-DM-3HHQ,which indicated that mpdG is likely involved in the cleavage of 2,6-DM-3HHQ.In conclusion,a 2,6-DMP-degrading bacterium M.neuroarum B5-4 was isolated and identified,and a metabolic pathway of 2,6-DMP was revealed.The mpdACDEFB cluster was proved to be involved in 2,6-DMP degradation.MpdAB was responsible for the initial degradation of 2,6-DMP and catalyzed the para-hydroxylation of 2,6-DMP to 2,6-DMHQ;MpdAB could also be used to hydroxylate 2,3,6-TMP to produce 2,3,5-TMHQ,a precursor of vitamin E synthesis.Maleylpyruvate isomerase MpdD and MpdAB were responsible for hydroxylation of 2,6-DMHQ to 2,6-DM-3HHQ.In addition,the functions of repressor regulatory protein MpdR and dioxygenase MpdG were studied preliminarily.The above results provided a solid foundation for the better use of microorganisms to remove 2,6-DMP pollutants in the environment.Furthermore,the mechanism of the second step hydroxylation of 2,6-DMP catalyzed by MpdD and MpdAB,and the transcriptional regulation mechanism of MpdR need to be further studied. |
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