| Cyclohexylamine is a flammable,toxic organic pollutants.During the process of production and usage,the released exhaust gas has a great harm on environment and human body.As we know,microbial degradation of organic pollutant is lowcost and efficient.So finding a strain which can degrade cyclohexylamine and studing the degradation mechanism is important.According to reports,there are only two pure cultures which could take advantage of cyclohexylamine as the sole carbon and nitrogen sources.One is a gram-positive bacteria Brevibacterium oxydans IH-35 A which isolated by Iwaki H.Another is a gram-negative strain Pseudomonas plecoglossicida NyZ12,screened and separated by the research group from Wuhan Institute of Virology Chinese Academy of Science.We obtained the whole genome information of NyZ12 by complete genome sequencing.Through comparative analysis and aligment,five genes,amo425,amo2631,amo4207,amo4637 and amo5539 were predicted to be candidates related to the degradation process of cyclohexylamine.In order to identify whether these genes are involved in the metabolism of cyclohexylamine from genetic respects,we knocked out five genes respectively,then analyzed gene functions by observing the characteristic changes of the mutants against the wild strain.Specific steps in construction of unmarkered knockout mutant are as follows:1.Amplification of homologous fragments:Designing appropriate primers to amplify homologous fragments of the target gene,then the upstream and downstream flanking DNA fragments of the target gene were fused by overlapping PCR.2.Construction of knockout vectors:The fusion fragment was connected to pGEM-T Easy and transformed into DH5α,identified by restriction enzyme digestion and sequencing.Then the fusion fragment released from the recombinant T vector and the suicide vector were double digested with corresponding enzymes and connected,then transformed into DH5α strains and identified.3.Conjugation:The recombinant suicide plasmid was introduced into E.coli S17lpir strains and transferred into NyZ12 by conjugation.Finally the recombinant suicide vector which containing homologous fragments recombined with genome of wild bacterial.4.Screening and characterization of mutants:Mutants were screened with antibiotic resistance and PCR,then the growth of gene-knockout mutants grown in cyclohexane inorganic salt medium was observed.Two methods of gene knockout were adopted in this study,insert knockout and unmarkered knockout.It is easy for insert knockout to screen the mutant by inserting the antibiotic gene in the target gene.However,insertion of antibiotic also may be a burden which led to the slow growth of mutants,after all,we chosed unmarked deletion.After unmarked knockout of the five genes,the results showed no significant effect on the growth of mutants.We proposed multiple gene knockouts of these amine oxidase genes in the follow-up study after considering the metabolic pathway of cyclohexylamine may be involved in multiple genes in NyZ12.We found five genes orf2866-2870 encoding the catalyzation of cyclohexanol or cyclohexanone to adipic acid were in an operon,and their expression level increased after the induction of substrate with transcriptome analysis.The expression level of each single gene was further confirmed by quantitative real-time PCR.In order to verify the function of cyclohexanone degradation gene chnB(orf2867),we knocked out chnB successfully.The mutant could not grow in inorganic salt medium which containing cyclohexylamine or cyclohexanone,which meant the degradation of cyclohexylamine via cyclohexanone in NyZ12.Finally the mutant recovered its growth ability after complementation test. |
