Novel approaches to improve functional screening of biocatalysts in metagenomic libraries

Abstract

Biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Currently, there is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in microbial genomes found in environmental samples and provides a set of new technologies directed towards screening of new catalytic activities with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal metagenomic screening strategies generally results in an extremely low success rate for enzymes identification. In this sense, the present proposal aims to perform three main approaches in order to develop new tools to improve target gene recovery. Firstly, two different strategies will be used to overcome the biased expression of foreign proteins in the screenings: (i) Construction of metagenomic libraries both in Pseudomonas putida and E. coli and; (ii) Utilization of the optimized and synthetic pSEVA broad host-range vector for library production. At present, there is no reported work in literature using a vector with similar features in functional metagenomic screenings. On the other hand, a third strategy comprising the engineering of a bacterial host will be developed using a synthetic biology approach. In particular, the strategy aims to improve the recovery rate of enzymes by exploiting the synergistic interaction between the metagenomic enzyme (or other accessory/complementary protein) and an enzyme expressed from the chromosome of the engineered host strain. Together, these approaches would provide a new, high efficient platform for the construction and functional screening of metagenomic libraries. (AU)