Investigation of genes involved in biodegradation of aromatic hydrocarbons from metagenomic mangrove impacted with petroleum

Abstract

Due to the great interest of the scientific community to discover new genes, metabolic pathways and bioactive compounds with biotechnological applications, be it in industry, in medicine, in their own academic field or even in the home society, began a frantic search these resources environments hitherto little explored. Based on this assumption, mangrove sediment becomes a significant source of new microbiological and genetic resources to be investigated. The mangrove ecosystem is a very peculiar and very important for the maintenance of life in the seas and the balance of the biosphere. It can be found along the mainland coast in estuarine areas, river deltas, lagoons and islands and is home to a multitude of living organisms such as fish, crustaceans, mollusks, birds, reptiles, mammals and various microorganisms. Microorganisms that live in this biome have key role in its maintenance as part of biogeochemical processes of the cycling of carbon and nutrients. By presenting extreme conditions such as high salinity, high temperatures, large variations in the level of the tides, much sedimentation and anaerobic soils, the possibility of finding extremoenzimas is much higher, which also serves as a justification for their studies. A lot of time these environments have suffered the impact of anthropogenic activities, especially with regard to disasters caused by oil spills. Oil is a complex mixture of organic compounds, including aromatic hydrocarbons, which, on contact with the mangrove sediment, encrusted remains long, endangering its biological diversity. Many microorganisms can degrade these compounds, further studies in this direction are being made to develop new approaches to bioremediation of contaminated environments, leveraging the metabolic potential of the same. Based on this information, this study aims to identify and characterize genes and enzymes that degrade aromatic dioxygenases from a library of clones fosmidiais metagenomics Mangrove highly impacted by oil, previously constructed and sequenced. To this end, the sequence data 454 by the platform (Roche) are processed in bioinformatics tools to locate groups of sequences for dioxygenases pathways involved in biodegradation of aromatic hydrocarbons. After being found and analyzed, specific primers will be designed for screening via PCR positive clones in the metagenomic library containing these genes. The DNA fosmidial positive clones will be extracted and sent to the pyrosequencing through outsourced service. The sequencing reads are assembled into contigs to establish the original DNA fragment followed by structural characterization of operons pathways for degradation of aromatic hydrocarbons. Specific primers are designed to amplify each gene individually contained in operons. These genes are cloned into expression vector (pET) for purification and molecular characterization of proteins involved in the biodegradation of aromatic hydrocarbons.