As the development of metagenomic technologies in the past ten years, it is unquestionned that microorganisms encompass the largest resource of metabolic and genetic diversity in the world. Actually, one gramme of soil contains more than 109 bacteria and 103-104 species. Some of their members are able to carry out enzymatic reactions leading to the complete degradation of pollutants (such as quinoline). So, the biodegradation of some highly toxic or organic compounds by microorganisms will be a general trend for pollutant treatment. However, the huge reservoir of molecules and enzymes from microorganisms still need to be explored because more than 99% of microorganisms cannot be cultivated in vitro.My work was based on collaboration between the University SJTU and Ecole Centrale de Lyon. Our partners at the University SJTU have built a laboratory scale denitrification reactor which was capable of degrading quinoline by removing the chemical oxygen demand. A new tool called "Genefish" has been developed in our laboratory as an alternative method for metagenomics which aims to discover novel industrial or environmental genes of interest. Following the early work in our laboratory, my thesis is presented here in two parts.In the first part, we set up a quinoline microcosm experiment both under aerobic and anaerobic condition using reference soil extensively studied in the laboratory at Ecole Centrale de LYON. This work aimed to reveal the potential bacterial diversity and even genes responsible for quinoline degradation. We used RISA(Ribosomal intergenic Spacer analysis) to analyze the bacteria community structure changes and GC/MS (Gas Chromatography-Mass Spectrometry) was also used to detect the quinoline degradation and reveal potential quinoline metabolic pathways under aerobic and anaerobic condition. Results showed great bacteria community structure changes and high quinoline degradation activity after the quinoline addition under aerobic condition. The future work is to investigate the bacteria community which may be responsible for quinoline degradation using the technique of NGS (Next Generation Sequencing).The second object of my thesis was to use the Genefish tool to capture targeted genes (the bcr gene responsible for the quinoline degradation in the wastewater treatment bioreactor) from the soil metagenome. The aim was to construct an E.coli strain containing a capture plasmid and Red system for capturing targeted genes from metagenomic DNA by homologous recombination. The capture plasmid includes a toxic cassette consisting of two suicide genes which can be activated by chemical induction, finally support the positive recombinants selection. It also contains two multiply cloning sites in which highly conserved sequences were inserted and works as the bait during recombination. We have tested the capacity of Genefish to capture the PCR products of bcr gene; the efficiency was low because of the persistence of several copies of the capture plasmid into the Genefish strain after recombination events. So, three strategies were tried to improve the recombination efficiency: co-electroporation, plasmid segregation and mono-copy capture plasmid construction. Finally, the strategy of plasmid segregation works but the recombination efficiency was still low maybe caused by the uncertain model of homologous recombination. The further research will focus on the transfer of the toxic cassette and homologous arms into the host strain chromosome, this new strategy will exclude the bad effect of low copy number capture plasmid, uncertain model of λ Red induced homologous recombination and the homologous arms site in the capture plasmid which are the most important factors influencing the homologous recombination efficiency in Genefish.
