Assessing and characterising the repertoire of constitutive promoter elements in soil metagenomic libraries inEscherichia coli

Abstract

SUMMARYAlthough functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we have developed a novel framework for prospecting, characterising and estimating the accessibility of promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Using the expression profiling of fluorescent clones from two independent libraries from soil samples, we directly analysed the regulatory dynamics of novel promoter elements, addressing the relationship between the “metaconstitutome” of a bacterial community and its environmental context. Through the construction and screening of plasmid-based metagenomic libraries followed byin silicoanalyses, we were able to provide both (i) a consensus exogenous promoter elements recognizable byEscherichia coliand (ii) an estimation of the accessible promoter sequences in a metagenomic library, which was close to 1% of the whole set of available promoters. The results presented here should provide new directions for the exploration through functional metagenomics of novel regulatory sequences in bacteria, which could expand the Synthetic Biology toolbox for novel biotechnological and biomedical applications.