Degradation Pathway Of M-Cresol In Comamonasthiooxydans CHJ601

Alkylphenol is a kind of aromaticity produced by the alkylation of phenol.Most of it comes from coal gasification,petroleum refining,and the chemical and petrochemical industries.m-cresol(3-methylphenol)is one of the most important alkyl phenol pollutants with high toxicity and potential carcinogenicity.Due to the relative stability of its structure caused by substituting methyl groups,it is difficult to be degraded in nature.It is necessary to screen out a strain thatcan degrade m-cresol efficiently to deal with environmental problems.In this study,13 strains of bacteria were isolated from sewage sludge collected from Yuekou Industrial Park in Tianmen,Hubei Province,and screened with m-cresol as substrate.The fastest-growing strain was obtained.The colony was found to be white and moist with a diameter of about 0.5-1.0 mm.The strain was identified as Comamonassp.by 16S r RNA gene,and we named it CHJ601.The final concentration of m-cresol was 2m M,and OD600 was 0.22 in 108h.CHJ601 was resistant to ampicillin,kanamycins,nalidixic acid,streptomycin and gentamicin,but not chloramphenicol and tetracycline.From the substrate growth experiments,strain CHJ601 could grow well in phenol,m-cresol,o-cresol,p-cresol,gentisate,protocatechuate,catechol,3-hydroxybenzoate and 4-hydroxybenzoate,but in all six xylenols can not grow.The inducible analysis showed that the expression of genes required for the degradation and metabolism of m-cresol in CHJ601 was not induced by the substrate m-cresol.Finally,we evaluated the bioremediation ability of the CHJ601 strain by zebrafish survival test.The results showed that the water treated with 0.1 m M phenol,m-cresol,o-cresol,and p-cresol by CHJ601 strain was better than that treated with 0.1 m M phenol,m-cresol,o-cresol,and p-cresol,after 24 hours,all zebrafish could survive.These results suggest that the CHJ601 strain has good prospects for bioremediation.The whole-cell biotransformation of CHJ601 was carried out with o-cresol,m-cresol,and p-cresol as substrates,respectively.HPLC and LC/MS analyzed the samples,and the intermediates of the intact m-cresol metabolism pathway and some o-cresol and p-cresol metabolism intermediates can be identified.m-cresol was first oxidized to 3-hydroxybenzyl alcohol,then to 3-hydroxybenzaldehyde,and finally to open-loop metabolism through 3-hydroxybenzoate.o-cresol enters the open metabolism directly through 3-methylcatechol.In addition,p-cresol becomes 4-hydroxybenzaldehyde and then becomes 4-hydroxybenzoate,which goes into ring-opening metabolism.CHJ601 strain was whole genome sequencing and functionally annotated based on the third-generation sequencing Pac Bio RS II technology platform.Theresults showed that the strain CHJ601 had only one chromosome and no plasmid;the genus of the strain was determined by constructing a genome-wide evolutionary tree,which was named Comamonasthiooxydans CHJ601.Genome annotation analysis revealed genes related to the complete phenol metabolism pathway;genes related to o-cresol and p-cresol metabolism could also be found;and by comparing genes related to p-cresol metabolism,it was found that mch AB(GEN04768-70)and mch C(GEN04771)may be involved in the metabolism of m-cresol.mch A(GE04768)has 79.11%identity with the xanthin subunit of p-cresol methylhydroxylase encoded bypch F gene in Pseudomonas putida NCIMB 9866.mch B(GE04770)had 45.65%identity with the cytochrome p-cresol methyl hydroxylase subunits encoded by pch C gene in strain 9866.mch C(GE04771)had 38.05%identity with the pch A gene encoding p-hydroxybenzaldehyde dehydrogenase from strain 9866.The function of the predicted gene was verified by heterologous expression,molecular docking,and product identification.The mch Aand mch B genes encode a two-component m-cresol methylhydroxylase,which catalyzes o-cresol,m-cresol,and p-cresol in the presence of NADH and FAD.The determination of kinetic parameters showed that m-cresol was the best substrate of the enzyme.The enzyme could catalyze m-cresol to produce 3-hydroxybenzyl alcohol and then oxidize to 3-hydroxybenzaldehyde.mch C gene encodes 3-hydroxybenzaldehyde dehydrogenase,which catalyzes the synthesis of 3-hydroxybenzaldehyde into 3-hydroxybenzoate in the presence of NAD~+.In addition,p-cresol methyl hydroxylase(Pch CF)and m-cresol methyl hydroxylase(Mch AB)were docked with m-cresol,respectively.It is suggested that m-cresol methyl hydroxylase differs from the previously reported enzyme.These results indicatethat the catabolism of m-cresol is preferentially completed by the methyl oxidation pathway in CHJ601 strain.In summary,a strain CHJ601 with m-cresol as the sole carbon source was screened,and its growth characteristics were studied.The complete degradation pathway of m-cresol and the partial degradation pathway of o-cresol and p-cresol were identified by HPLC and LC/MS,mch AB(GEN04768-70)encodes a novel m-cresol methylhydroxylase,which catalyzes the synthesis of 3-hydroxybenzyl alcohol from m-cresol and the oxidation of m-cresol to 3-hydroxybenzaldehyde mch C(GEN04771)gene encodes 3-hydroxybenzaldehyde dehydrogenase,which catalyzes the conversion of 3-hydroxybenzaldehyde to 3-hydroxybenzoate.Finally,CHJ601 has the prospect of bioremediation provedby zebrafish.