Screening And Characterization Of Aromatic Oxygenase Based On Metagenomic Technology

It is estimated that only less than 1% microbes are cultivable in laboratory using pure culture technology due to different growth requirements, thus limiting our understanding of microbial diversity. Metagenomic technology without cultivation opens a new way to access the uncultured microbes based on direct isolating total DNA from environment samples. In this dissertation, aromatic oxygenases were screened from activated sludge by different metagenomic strategies, and the discovery strategies were evaluated in detail.Four methods were used to extract large-fragment metagenomic DNA from activated sludge for library construction, including lysozyme (L)-SDS-proteinase K method, TaKaRa Kit, CTAB method and agarose-embedding method. Lysozyme (L)-SDS-proteinase K method was optimized for DNA extraction using single-factor method and response surface methodology, and several key factors was identified. The optimal conditions for DNA extraction were as follows:TENC as extraction buffer,1.5% of CTAB,0.5 mg/mL of lysozyme, water bath at 37℃for 1.4 h,2% of SDS,200μg/mL of proteinase K, water bath at 55℃for 2.5 h, DNA precipitation for 40 min with isopropanol. The highest DNA yield was obtaind with 170μg per gram sludge under the optimal conditions. However, the DNA fragment was not large enough for library construction obtained from the first three methods. By contrasted, agarose-embedding method was used to extract larger than 1.9 Mbp metagenomic DNA, which could lysis cell in situ and promise less DNA shearing. Fosmid library was generated from the metagenomic DNA, and the library contained 5,280 clonies, with an average insert size of approximately 35 to 40 kb. Therefore, it was estimated that there was approximately 200 Mb of metagenome DNA in total. And restriction analysis revealed the DNA fragments in library presented a high level of diversity.Three metagenomic strategies, i.e., direct PCR, function and sequnce screening, were used for screening aromatic ring-hydroxylating and ring-cleaving oxygenases from the metagenome. However, no aromatic oxygenase was identified from Fosmid library by function screening, which was possibly due to the unsuccessful heterologous expression of multicomponent oxygenase in E. coli. Then an effective PCR-screened method was designed to obtain a positive clone (designated pCC3B) from the library. A 206 bp amplicon from clone pCC3B was confirmed as the conserved region of phenol hydroxylase gene. In addition, the partial gene (719 bp) of 2,3-dihydroxybiphenyl 1,2-dioxygenase was obtained from the metagenomic DNA by direct PCR method.Genome walking including TAIL-PCR, reverse PCR and Clontech Kit, were used to clone the flanking region, and resulted in 4,760 bp full length of phenol hydroxylase gene. And the gene sequence has been submitted to GenBank database with the number HQ334893. The sequence analysis showed the KLMNOP components share 44-77% amino acid identity with the known components. In addition, phylogenetic tree suggested that this gene belogngs to group I with low Ks value, which is the dominant gene in sewage treatment system, whereas the phenol hydroxylases from pure culture often process higher Ks value. It was exhibited that metagenomic technology was potential for accessing to the novel and dominant functional genes.Four bases of phenol hydroxylase gene mutated when the gene was ligated into vector pET28a (+). Then the phenol hydroxylase gene after modification was transformed into E. coli for expression. The results showed that not all of six components were expressed, and most protein was insoluble inclusion body, so that no activity of phenol hydroxylase was detected. There was no increase of enzyme activity even though phenol hydroxylase was expressed at lower temperature. In addition, it was found that the promoter and ribosome-binding site (RBS) of phenol hydroxylase gene was diffrent with those from pure cultures. Therefore, it was inferred that partial expression, inclusion body and novel gene organization should lead to the loss of phenol hydroxylase activity.TAIL-PCR was used to amplify the 3’-end unknown region of 2,3-dihydroxybiphenyl 1,2-dioxygenase gene, and the full length with 897 bp was obtained (designated bphC_meta). The protein (BphC_meta) shared 99% sequence identity with that of Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400. The phylogenetic relationship indicated that BphC_meta was classified as a member of subfamilyⅠ.3.A, which belongs to type I extradiol dioxygenases group. A base of bphC_meta also mutated while it was ligated into vector pET28a (+). After medication, bphC_meta was successfully expressed in E. coli. The experiment of metal-ion dependence suggested that BphC_meta is Fe (Ⅱ)-dependent dioxygenase. BphC_meta was successfully purified from recombinant E. coli with a subunit molecular mass of 32±1 kDa. The kinetic analysis indicated that enzyme reaction of BphC_meta consisted with substrate-inhibitory models, and BphC_meta preferred substituted catechols in the order 2,3-dihydroxybiphenyl> 3-methylcatechol> catechol. However, no activity for 4-methylcatechol and 4-chlorocatechol was determined. The optimal pH and temperature for BphC_meta was 9.0 and 40℃using 2,3-dihydroxybiphenyl as substrate. In order to further explain the relationship between BphC_meta structure and activity, five substrates mentioned above were successfully docked into the active sites of BphC_meta. And the affinity of BphC_meta predicted by docking was consistent well with the kinetic data. Docking studies suggested that the position and type of substituents for catechol resulted in different Fe-O distance between enzyme and substrate, which produce different performace of BphC_meta for substituted catechols.Taking the example of aromatic oxygenase, three metagenomic strategies and culture-dependent technology were evaluated by analyzing the time consume, cost, gene novelty and expression feasibility of aromatic oxygenase. This study will provide novel biocatalysts for bioremediation and chemical industry, and promise a research model for exploitation of gene and enzyme resources from uncultivable microbes.