AZOZYMES: A metagenomic study to identify enzymes of bacterial origin with biotechnological interest from Azorean marine extreme environments.

Objectivos

1. Sample collection from unconventional marine environments
   – Several scientific cruises planned for a near future to the hydrothermal vent sites in the vicinity of the Azores, the Lucky Strike and Menez Gwen sites, will be intend for animal and microbial samples collecting. Technology and diving gears will rely greatly on the Portuguese ROV “Luso” and the crew from the Gago Coutinho Navy vessel commissioned by the Portuguese Hydrographic Institute.
   – It is planned to continuing the bio-prospection of the shallow hydrothermal waters at the D. João de Castro Bank, between the island of Terceira and São Miguel, and the vent site “Espalamaca” located off the bay of Horta and collecting various samples of microbial mats. A number of bacterial mat samples have already been collected at the bottom of hydrothermal vents or on the surface of active vent chimneys and have been preserved in ethanol to be analyzed in a near future.
2. To assess the community composition and structure of marine bacteria populations under study, by a direct molecular approach.
   Several PCR primer sets are already available for the molecular detection of bacterial chitinase genes in terrestrial and aquatic ecosystems. However, there is scarce information related to specific primers for PCR amplification of chitinase gene fragments in Achaea domain. These primers shall be designed by alignment and selection of highly conserved regions of selected chitinase sequences from the GenBank database. PCR products will be analysed and related sequences will be compared with databases and identified by homology searching. To confirm identification, phylogenetic trees will then be constructed, including the most homologous and related sequences found in the databases. A chitinolytic community TGGE profiles database from hydrothermal vent sites sampled will be kept.
   Screening of different proteolytic bacteria may lead to the possible identification and subsequent cloning of diverse proteinase genes that may have, for instance, enhanced chitinolytic properties.
3. Construction of metagenomic libraries using advanced technology, modern sequencing, sequence annotation and cutting-edge bioinformatics resources.
   With the aim of discovering extremophile enzymes from microorganisms of three distinct marine habitats, a DNA sequence-based approach using metagenomic analyzes will be done, that it sequencing all the genomes present in microbial communities. Metagenomic studies and transcriptome analyses will be performed at the Biotech Park, Biocant, and “deep” sequencing such as the 454-sequencing or RNAseq from Ilumina technology will generate several thousands of DNA sequences that will be stored in the database created for this unique purpose. Bioinformatics resources and tools developed in an attempt to maximize the capacity to analyze these vast datasets will support advanced data acquisition, data storage, data management, data integration, data mining, data visualization and other computing and information processing services over the Internet in a so-called e-infrastructure.
   The provision of dedicated web-based resources and e-infrastructures is essential for advanced research in marine ecology and biotechnology. The identification of new genes of interest, most likely, genes encoding extremophile enzymes with putative biological functions may be biotechnology relevant.
4. Genetic engineering technologies. Promising genes will then be cloned and expressed in synthetic systems, or heterologous expression systems, to assess their biological activity in vitro systems. Synthetic enzymes should be tested for their biological activity and potential use in biotechnology. Some of the enzymes planned to be targeted have a direct implication in industry: proteases, amylases, pullulanase, glucoamylases, glucosidases, cellulases, xylanases¸ lipases, esterases. Potential new “Azorean” marine enzyme candidates are: chitinase, alpha xylanase, xylose isomerase. Candidate enzymes will be further assessed for their commercial value and patenting.
   There is a need of selection of potentially useful enzymes during the screening process. Generated bioproducts must attempt to the region’s sustainability. With its wealth of agricultural and water resources, the Azores are well-positioned to improve country’s strategic interest in clean technologies and bring substantial benefits to the region including cost-effective biotechnology products and optimal designs for remediation strategies.