Researches On The Molecular Mechanism Of Dibenzofuran Degradation By Microorganisms

Dibenzofuran?DBF?,as a model dioxin-like compound,is toxic,carcinogenic,teratogenic,mutagenic and persistent environmental pollutants.Microbial degradation of DBF can be categorized into two main pathways:bacteria using DBF as the sole carbon source to grow can conduct degradation via an angular dioxygenation pathway;microorganisms can co-metabolize DBF via lateral dioxygenation after cultivated with biphenyl or naphthalene.Compared with the angular dioxygenation pathway,the lateral dioxygenation pathway has not been revealed clearly.Although some new downstream metabolites of lateral dioxygenation pathway have been identified,but the entire pathway and enzymes involved remain unclear.In this study,whole cells of Pseudomonas putida strain B6-2 was used to degrade DBF and accumulate 2-hydroxy-4-?3'-oxo-3'H-benzofuran-2'-yliden?but-2-enoic acid?HOBB?via lateral dioxygenation pathway.After optimization of media,pH,cell density,DBF concentration,extraction method and extractants,bacteria was first cultivated in LB medium containing biphenyl,then a batch process in which cells were used twice was conducted with an intial DBF concentration of 0.5 mM at pH 7,OD₆₀₀=5.After extraction with ethyl acetate via the neutral-acid method,13.58±0.31 mg of HOBB was obtained from 22.49±0.74 mg DBF with a high overall production yield of 60.4%?w/w?.The purified HOBB,which is a yellow powder,could be detected and identified by LC-MS,GC-MS and NMR.In order to find the genes involved in the HOBB degradation pathway,firstly by comparing the degradation pathway of biphenyl,it is speculated that vinylpyruvate hydratase?VPH?can catalyze the degradation of HOBB.The related genes in strain B6-2 were knocked out and were expressed heterologously in E.coli respectively,proving that the VPH was not involved in HOBB degradation.Subsequently,a molecular structure of an intermediate 4-[1-hydroxy?2-naphthyl?]-2-oxobut-3-enoic acid is similar to HOBB in the biodegradation pathway of phenanthrene,and PhdG was involved in its degradation.Functional verification of a recombinant E.coli has proved that PhdG can participate in the degradation of HOBB.Using pET28a to express the protein,the crude enzyme reaction showed a weak degradation to HOBB;and the conditions of expression and purification should be optimized.In order to furtherly verify the function of PhdG,we successfully integrated the phdG gene into the strain B6-2 genome.Then function verification should be followed.In this study,we accumulated and prepared the intermediates HOBB in DBF lateral degradation pathway.Then function verification,protein expression and purification were performed with the predicted genes of HOBB degradation.The research works on mechanism of the lateral dioxygenation pathway,and the search for the exogenous genes of HOBB degradation has provided new ideas to accelerate the degradation rate of DBF.