The Molecular Mechanism Of Degradation Of Tetrahydrofuran By Pseudomonas Oleovorans .DT4

Tetrahydrofuran(THF)is an important raw material for organic synthesis and is an excellent solvent.Its extensive use and emissions in industrial production have brought great harms and impacts to the ecological environment and human health.Therefore,the application of highly efficient and environmentally friendly biodegradation of THF is a significant act.The studies of this paper were carried on the THF-degrading bacteria DT4,focusing on the degradation mechanism of THF at genetic level,which can provide theoretical basis and reference for application of THF biodegradation.The functions of plasmids of DT4 during THF degradation and their relation with antibiotic resistance of DT4 were studied using plasmid eliminated strains.The optimum conditions for plasmid elimination of DT4 as follow: treating the cells with 40 ℃-SDS method,and then repeat for 5 times.The results showed that THF degradation of DT4 declined after the elimination,and its resistance to chloramphenicol(Cm)and tetracycline(Ter)disappeared,indicating that the plasmids of DT4 contained THF degradation-related genes and antibiotic resistance genes of Cm and Ter.The whole genome of DT4 was sequenced by Illumina HiSeq2000 high throughput sequencing platform.The results showed that the genome size was 4.6 Mb and the GC content was 62.30%.Bioinformatics analysis showed that the strain contained 4317 ORFs,62 tRNAs and 49 sRNAs.The gene information and function of strain DT4 were analyzed by analysis of common databases such as RAST,COG,GO and KEGG.THF degradation related genes of DT4 were studied through transposon mutagenesis.The optimal conditions for DT4 to be mutated by transposon pSC123 were as follow: with the mixing ratio of 1: 1,followed by a conjugation process for 24 h,and incubate at 30℃ for 3 d,a mutation efficiency of 0.416% could be achieved.After several screening,a total of 137 mutants was obtained,one of which named Mut12 was chosen for studies that followed.A 5036 bp sequence was obtained by SEFA-PCR.After analysis,three genes,orf A,orfB and orf C,were found to encode aldehyde dehydrogenase,esterase family protein and cyclohexanone monooxygenase respectively.The transposon insertion site was located between 637-638 bp of orfB gene.The esterase protein encoded by the orfB gene is responsible for the hydrolysis of the esters material of the γ-butyrolactone,a THF degradation intermediate,and is involved in the intermediate process associated with THF degradation.The effect of orfB on the degradation of THF was confirmed by the recovery of the orfB gene to the mutant Mut12,which indicated that orfB was critical to THF degradation.