Conversion of lignocellulosic biomass into biofuels is a promissory alternative to replace fossil fuels derived from nonrenewable energy sources. Lignocellulose consists primarily of cellulose, hemicellulose and lignin. Cellulose is the most recalcitrant material present in plant cell walls, but has a substantial potential as a bioethanol production source. Currently, industry requires enzymes with tolerance to process-specific parameters or improved catalytic performance. In this way, metagenomics allows the identification of new biocatalysts with specific activities without the need of previous isolation and cultivation of microorganisms. Several examples reported in literature showed the identification of new enzymes and other bio-molecules from different environments through functional metagenomic screening. However, the discovery of new genes by metagenomic approaches has been limited. The probability of identifying a target gene depends on many factors that are intrinsically linked. For instance, the efficiency of heterologous gene expression of a target gene from a metagenomic DNA fragment is restricted by the molecular machinery of the heterologous host organism employed in the screenings and the use of non-optimized vectors resulting in low enzyme identification rates. Previous studies suggest that about 40% of the enzymatic activities may be readily recovered by random cloning in Escherichia coli, the most frequently bacteria used for functional metagenomic screenings. In order to circumvent these limitations, the present proposal aims to develop new tools to improve target gene recovery in functional metagenomic screenings. For this, it is proposed (i) construct metagenomic libraries in broad-host-range vectors (pSEVA) and (ii) developof a new vector pointing to improve the screening strategy, the pCELsyn vector which is derived from a pSEVA vector. In this sense, pCELsyn plasmid harboring the Cel5A endoglucanase from Bacillus subtilis will be used in synergistic screenings in order to increase celullase activities identification. The screenings will be performed in two hosts: E. coli and Pseudomonas putida, aiming to sort out the insufficient or biased expression of foreign genes in E. coli, as described by several studies. The metagenomic libraries will be generated from biomass-rich soil samples since it probably will be enriched in genes encoding for cellulases. Functional screenings will be perform using cellulase screening methodologies well-established in literature. Thus, it is expected to improve the efficiency of enzyme recovery in metagenomic screenings.
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